Aqueous dynamics in exfoliation syndrome.

Eighteen untreated patients with unilateral exfoliation syndrome without glaucoma were studied with regard to differences in aqueous dynamics between the affected and unaffected fellow eyes. Additionally, both eyes of the patients with exfoliation were compared to the eyes of 18 age-matched and gender-matched control subjects. Intraocular pressure was significantly higher (13.9 +/- 3.0 mm Hg [mean +/- standard deviation]) in the affected compared with the unaffected eyes (12.2 +/- 2.5 mm Hg), but no difference in aqueous humor flow was detected when eyes in the three groups were compared (affected eyes, 2.40 +/- 0.60 microliter/min; unaffected eyes, 2.40 +/- 0.65 microliter/min; control eyes, 2.61 +/- 0.60 microliter/min). Anterior chamber volumes were significantly smaller in affected (122 +/- 37 microliters) and unaffected eyes (121 +/- 37 microliters) of the patients with exfoliation compared with the eyes of the control subjects (145 +/- 30 microliters). Apparent resistance was higher in the eyes with exfoliation (6.5 +/- 1.9 min mm Hg/microliters) compared with both unaffected (5.4 +/- 1.2 min mm Hg/microliters) and control groups (5.2 +/- 1.8 min mm Hg/microliters), although these differences were not statistically significant.


Intraocular pressure decrease in household contacts of patients with Hansen's disease and endemic control subjects.

We compared the intraocular pressure in 150 urban household contacts of patients with Hansen's disease and 132 endemic control subjects from an urban population in Karachi, Pakistan, who were matched in regard to race, age, gender, and socioeconomic status. The mean intraocular pressure in the upright position was 12.6 mm Hg in the right eye and 12.9 mm Hg in the left eye in household contacts of patients with Hansen's disease and 15.3 mm Hg in the right eye and 15.4 mm Hg in the left eye in the endemic control population (P < .0005). The postural change in intraocular pressure from the upright to supine position was 1.7 mm Hg in the right eye and 2.1 mm Hg in the left eye in the household contacts group and 1.3 mm Hg in the right eye and 1.5 mm Hg in the left eye in the endemic control group (P < .0006). Our findings, especially in view of past observations in intraocular pressure changes in patients with Hansen's disease, raise serious questions about possible early ocular susceptibility to Mycobacterium leprae infection and about public health issues, including possible early indication of disease.


Asymmetric pigmentary glaucoma resulting from cataract formation.

Pigment dispersion syndrome usually manifests bilaterally, and asymmetric involvement is unusual. When asymmetry is present, the eye with greater involvement may have an additional exacerbating condition or the eye with less involvement may be protected. Analysis of such cases should further elucidate the mechanism of the disorder and its development and regression. We examined four patients in whom unilateral cataract formation or extraction was associated with reduced clinical signs of pigment dispersion syndrome in the affected eye. Cataract formation, by inducing relative pupillary block, appears to decrease or prevent the manifestation of pigment liberation.


The edrophonium-Hess screen test in the diagnosis of ocular myasthenia gravis.

The interpretation of the edrophonium (Tensilon) test in the diagnosis of acquired strabismus caused by myasthenia gravis has been problematic because of poorly defined endpoints and unknown sensitivity and specificity. We evaluated the endpoint criteria, dynamics, sensitivity, and specificity of binocular alignment in response to edrophonium by using the Hess screen test in ten normal control subjects, 12 nonmyasthenic patients with acquired strabismus, and in ten patients with acquired strabismus caused by ocular myasthenia gravis. A positive response to the edrophonium-Hess screen test was defined as a 50% or greater reduction in the strabismic deviation at the fixation point associated with maximum deviation within one minute of edrophonium infusion. All myasthenic patients had a 50% or greater reduction in the initial deviation within one minute of edrophonium infusion. Myasthenic patients had a statistically significant reduction in the average deviation up to 150 seconds after edrophonium infusion (P < .05 for all time periods). In contrast, with or without edrophonium infusion, control subjects had a purely horizontal fluctuation in binocular alignment of less than or equal to 2 degrees for the entire four-minute period after edrophonium infusion. None of the 12 nonmyasthenic patients tested positive to the edrophonium-Hess screen test. According to the criterion of positive response as was defined in this study, the test had a high sensitivity and specificity in this sample. These results suggest that clearly defined endpoint criteria make the edrophonium-Hess screen test a sensitive and specific quantitative study for the diagnosis of acquired strabismus caused by myasthenia gravis.


A stem cell niche theory of intestinal crypt maintenance based on a study of somatic mutation in colonic mucosa.

In both large and small intestine, mutagen administration leads to the occurrence of isolated crypts that are completely populated by a mutated phenotype; therefore, it has been proposed that crypts are maintained by a single stem cell. We show in mice that a single dose of mutagen leads to an early transient increase in frequency of colonic crypts that show a partial mutated phenotype and a later increase in frequency of crypts that show a complete mutated phenotype. This increase reaches a plateau at about the same time as the disappearance of partially mutated crypts. The same is true in the small intestine, but the time course is much slower. We propose an explanation based on multiple crypt stem cells that occupy a "stem cell niche," with random cell loss after stem cell division. A small difference in the number of crypt stem cells that occupy the niche provides a simple explanation for the surprisingly large difference in the time course of phenotypic changes in the large and small intestines after administration of a single dose of mutagen.


A (CA)n dinucleotide repeat assay for evaluating loss of allelic heterozygosity in small and archival human brain tumor specimens.

Southern blotting is a widely used method of determining loss of chromosomal alleles in tumors, but cannot be used to analyze small biopsies and most fixed, embedded tissues. These problems preclude analysis of many surgical neuropathology specimens. We have employed a polymerase chain reaction assay for loss of heterozygosity (LOH) based on the (CA)n dinucleotide repeat polymorphisms found in abundance throughout the human genome. We compared this method to conventional Southern blotting in detecting LOH on chromosome 10 in gliomas. From tissue sections of 14 paraffin-embedded, formalin-fixed gliomas, we amplified the (CA)n repeat D10S89 locus and compared (CA)n repeat patterns between tumor DNA and constitutional DNA. Loss of one chromosome 10 (CA)n repeat allele was seen in informative glioblastomas that showed allelic loss by Southern blotting, but not in gliomas that had maintained both alleles. The (CA)n repeat method can be applied to small and fixed, embedded specimens, is rapid and simple to perform, and uses highly polymorphic probes. We suggest that (CA)n repeats are a less exclusive and more rapid means of studying LOH in brain tumors than Southern blotting, and will provide further mapping data for the identification of tumor suppressor genes integral to glial tumorigenesis.


Immunohistochemical identification of thrombospondin in normal human brain and in Alzheimer's disease.

Thrombospondin is part of a family of adhesive glycoproteins and is involved in a number of physiologic processes such as angiogenesis and neurite outgrowth. Immunohistochemical localization of thrombospondin in normal human brains was investigated in the hippocampus and inferior temporal cortex. Two antibodies (one polyclonal and one monoclonal) against thrombospondin-labeled microvessels, glial cells, and a subpopulation of pyramidal neurons. The distribution of thrombospondin staining in patients with Alzheimer's disease was found to be comparable to control subjects. However, in patients with Alzheimer's disease a subset of pyramidal neurons that may be vulnerable in Alzheimer's disease exhibited decreased staining. This decrease in the intensity of labeling might constitute a marker for a neuronal population prone to early degeneration. In addition, thrombospondin staining was demonstrated in senile plaques in Alzheimer's disease. These results suggest that thrombospondin may be involved in the process of neuronal degeneration and senile plaque formation.


The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1.

Nephrosis induced in rats by puromycin aminonucleoside treatment (PAN) results in the apical displacement of the glomerular filtration slit membrane by newly formed, intercellular occluding-type junctions. Similar changes can also be induced by acute kidney perfusion with protamine sulfate (PS). We have investigated the molecular nature of these altered junctions using an antibody to ZO-1, a protein found exclusively in tight junctions. Immunoblotting demonstrates ZO-1, a 225-kd band, in glomerular extracts of normal, PAN-, and PS-treated rats. By immunofluorescence, ZO-1 was localized at the base of podocytes outlining the capillary loops of glomeruli from all three experimental groups. At the electron microscope level, using immunoperoxidase or immunogold labeling, ZO-1 was concentrated along the cytoplasmic surfaces of the slit diaphragms of normal rats. In PAN or PS rats, it was concentrated along both the newly formed occluding-type junctions and the remaining slit diaphragms. When podocalyxin (the major membrane sialoprotein of the podocyte) was similarly localized, it was found exclusively apical to the displaced slit membrane. Based on morphology and the presence of ZO-1, the altered junctions seen in PAN and PS rats appear to represent bona fide tight junctions. Their rapid (15-minute) induction in PS-treated rats suggests that on neutralization of the cell surface charge by polycation perfusion, discontinuous tight junctions form from a preexisting pool of junctional proteins. These findings raise the possibility that glomerular hydraulic conductivity may be regulated in part by regulating the relative patency and width of the filtration slits through focal tight junction assembly.


Association of murine CD31 with transmigrating lymphocytes following antigenic stimulation.

Human CD31 is a recently characterized molecule present on leukocytes, platelets, and endothelium. Its function is not known. Because it is a member of the immunoglobulin superfamily and structurally homologous to carcinoembryonic antigen, a putative intercellular adhesion molecule, it is believed that CD31 may function also as an adhesion molecule. In this report, we characterize the cellular reactivity of a monoclonal antibody to a murine protein that is homologous to CD31. To delineate the cellular reactivity of the murine CD31 homologue recognized by our monoclonal antibody, we used immunoperoxidase and immunoelectron microscopic techniques. The most striking finding was that the putative murine homolog of CD31 is expressed in particularly high amounts on endothelium-adherent lymphocytes transmigrating across sinusoidal or venular vascular boundaries. Such a distribution was apparent in draining murine lymph nodes during the peak of an immune response after immunization with a protein antigen in adjuvant, a situation in which there are many transmigrating lymphocytes. Immunoelectron microscopic analysis also shows that CD31 is predominantly distributed on portions of transmigrating lymphocytes that are in contact with or adjacent to areas of contact with endothelial cells. These findings suggest a previously undescribed role for CD31 in lymphocyte recruitment and transmigration.


Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas.

Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.7 mm, but were distributed focally in elongate patches bounded both lumenally and medially by deposit-free tissue in thick atheromas. Saline extracts generally showed undegraded monomers and dimers by electrophoresis. The residual protein contained A alpha and gamma-chains that were cross-linked predominantly (>80%) into unresolved high M(r) (>200 kd) derivatives, whereas B beta-chains were left non-cross-linked, as occurs in late stages of cross-linking by transglutaminases. The resolved components had electrophoretic mobilities corresponding to characteristic products of both factor XIIIa and tissue-transglutaminase. A greater incorporation of alpha- rather than gamma-chains into cross-linked products implicated tissue-transglutaminase as contributing heavily. By contrast, vascular graft pseudo-intimas and a cadaveric clot were rich in degraded fibrin devoid of fibrinopeptide A, and cross-linked in patterns typical of XIIIa with gamma 2 dimers constituting the principal product. The findings indicate that the fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue-transglutaminase without being converted to fibrin.


The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues.

Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen.


Glomerular macrophages and the mesangial proliferative response in the experimental nephrotic syndrome.

Mesangial cell proliferation, which is a harbinger of glomerulosclerosis, occurs in both immune and nonimmune glomerulopathies. The proximity of infiltrating glomerular macrophages to the contractile mesangial cells during acute puromycin aminonucleoside (PA) nephrosis suggests the possibility of a paracrine effect on mesangial cell growth. To test this, three maneuvers to either raise or lower the glomerular macrophage number during acute PA nephrosis (2 weeks after PA) were employed: 1) an essential fatty acid-deficient (EFAD) diet; 2) a cholesterol-supplemented diet (CSD); and 3) a single dose (600 rad) whole-body X-irradiation (XI) given to CSD-fed PA rats. Both the glomerular macrophage number and proliferation within the mesangium were evaluated immunohistochemically with ED-1, a mouse monoclonal anti-rat macrophage label, and 19A2, a mouse monoclonal anti-proliferating cell nuclear antigen (PCNA)/cyclin antibody, respectively. Immunohistochemical detection of 5'-bromo-2'-deoxyuridine (BrdU) incorporation confirmed that proliferation was occurring within the mesangial zones. The EFAD diet significantly reduced both the glomerular macrophage and PCNA/cyclin-positive cell number at 2 weeks after PA with a positive correlation (r = 0.89, P < 0.05). The CSD maneuver significantly increased both the glomerular macrophage and PCNA/cyclin cell number with a strong degree of correlation (r = 0.95, P < 0.01). X-irradiation administered to CSD-fed PA rats significantly lowered both the glomerular macrophage and PCNA/cyclin-positive cell number at 2 weeks. In all groups, the glomerular tufts did not express muscle actin using HHF 35, a specific immunolabel, suggesting that the proliferation in this model is not related to direct mesangial cell injury. This study shows that maneuvers that modulate the glomerular macrophage number are also associated with corresponding changes in the number of proliferating cells within the mesangium, suggesting a paracrine growth stimulation by the infiltrating macrophage during acute PA nephrosis. The infiltrating glomerular macrophage may be an effector mechanism for the propagation of initial glomerular injury to glomerulosclerosis by augmenting mesangial cell proliferation early in the course of this nonimmune progressive glomerulopathy.


Morphologic effects of hGRH gene expression on the pituitary, liver, and pancreas of MT-hGRH transgenic mice. An in situ hybridization analysis.

Morphologic changes in the pituitary, liver, and pancreas of mice with the metallothionein-human growth hormone--releasing hormone (MT-hGRH) transgene were analyzed by in situ hybridization histochemistry (ISH). There was progression from somatotroph hyperplasia to neoplasia in pituitaries of transgenic mice. Pituitary neoplasms were present between 9 to 12 months of age in some mice. Magnetic resonance imaging (MRI) readily identified enlarged pituitaries in MT-hGRH transgenic mice. Serum mouse GH and hGRH levels were marked elevated in MT-hGRH transgenic mice. In situ hybridization histochemistry showed mRNA for hGRH in liver, pituitary, pancreas, spleen, and in most other tissues examined. Combined ISH and immunohistochemistry in the pituitary gland showed that some of the GH cells also produced hGRH, and ultrastructural immunohistochemical analysis of pituitaries showed that GH and hGRH were localized in the same cell and within the same secretory granules. Liver cells of MT-hGRH transgenic mice showed evidence of hypertrophy, and the pancreatic islets were hyperplastic with significant increases in the islet cell areas. The morphologic changes in the liver were distinctive enough to separate control littermates from MT-hGRH transgenic mice in all cases. The enlarged pancreatic islets had increased numbers of insulin-producing cells. Immunoreactive hGRH and hGRH mRNA were both localized in islet cells, and an intense hybridization signal of hGRH mRNA, but only weak staining for hGRH protein, were detected in the liver of transgenic mice. These results indicate that excessive hGRH production leads to distinct morphologic changes in various organs in MT-hGRH transgenic mice and that there is temporal progression from hyperplasia to adenomatous somatotrophs in pituitaries with chronic stimulation by hGRH that involves paracrine, endocrine, and autocrine mechanisms.


Functional heterogeneity and pathogenic significance of interleukin-6 in B-cell lymphomas.

A possible autocrine effect of interleukin-6 (IL-6) on the growth and differentiation of the tumor cells of 55 B-cell lymphomas was examined. Interleukin-6 was detected in a few types of B-cell lymphomas, including polymorphic immunocytoma (PI), small lymphocytic lymphoma (SLL), and immunoblastic lymphoma (IBL) with or without plasmacytoid differentiation. In PI and in IBL with plasmacytoid differentiation (IBL-P), IL-6 was detected only in immunoglobulin-containing plasmacytoid cells, and it was absent from most proliferating (Ki-67/PCNA-positive) lymphoma cells. In SLL, IL-6 was not observed in lymphoplasmacytoid cells; instead, IL-6 was observed in transformed (Ki-67/PCNA-positive) tumor cells in proliferation centers. The lymphoplasmacytoid cells in SLL exhibited a phenotype (IL-6/glutathione-S-transferase-pi [GST-pi]-negative), different from that of normal plasma cells (IL-6-negative/GST-pi-positive) and from the plasmacytoid cells (IL-6/GST-pi-positive) in PI and IBL-P. In IBL without obvious plasmacytoid differentiation, IL-6 was detected in most tumor cells that were highly proliferative (Ki-67/PCNA-positive). In this study, IL-6 was undetectable in most lymphomas related to follicular centers, in lymphoblastic lymphoma, in small noncleaved cell lymphomas of the Burkitt and non-Burkitt types, and in diffuse large cell lymphoma. This finding is compatible with a previous finding that IL-6 mRNA was absent from follicular center cells in reactive lymphoid tissues. The functions of IL-6 in these lymphomas may be quite diverse. It appears that IL-6, as an autocrine factor, is responsible for the plasmacytoid differentiation of lymphoma cells in IP and some IBL (IBL-P). The differentiation of lymphoplasmacytoid lymphoma cells in SLL, however, may not be mediated by an autocrine IL-6 mechanism. Interleukin-6 may provide a growth signal, rather than acting as a differentiation factor, for some IBL cells and for some transformed tumor cells in proliferation centers in SLL.


Early extracellular and cellular lipid deposits in aorta of cholesterol-fed rabbits.

Subendothelial accumulation of extracellular liposomes rich in unesterified cholesterol has been described as an early feature of atherosclerosis induced by cholesterol feeding in rabbits. Beta-very-low-density lipoproteins, however, the presumed source of atherogenic lipid in this animal model, contain mostly esterified cholesterol. The purpose of this study was to test for the presence of extracellular neutral lipid deposits consistent with esterified cholesterol, by employing new electron microscopic techniques. Rabbits were fed 0.5% cholesterol, 5% butter for 0, 1, 2, and 4 weeks. The lipid-preserving ultrastructural techniques showed, in control and atherosclerotic rabbit arteries, neutral lipid droplets adherent to the endothelial luminal surface. After 1 to 2 weeks, subendothelial extracellular deposits of mostly membranous lipid appeared; these deposits contained variable amounts of neutral lipid. At the same time, cytoplasmic neutral lipid droplets appeared in smooth muscle cells and in a small number of subendothelial macrophagelike cells. After 4 weeks, monocytic infiltration and macrophage foam cell development were prominent, but abundant extracellular lipid deposits also were found. Therefore, in arteries of cholesterol-fed rabbits, deposition of membranous and neutral lipid in the extracellular space and neutral lipid accumulation in resident arterial cells are early and probably independent events, both occurring before monocytic infiltration of the arterial intima.


Proto-oncogene analysis in multiple myeloma.

The genetic basis for the development of multiple myeloma (MM) remains poorly understood, in part because MM has thus far been relatively refractory to cytogenetic analysis. The few cases karyotyped have pointed to involvement of 11q13, site of the BCL1 proto-oncogene, or of 8q24, site of the MYC proto-oncogene. A recent molecular study detected rearrangements distal to the MYC gene in 16% of MM, using the MLVI-4 probe. The immunocytochemical demonstration of BCL2 protein overexpression in at least some cases of MM has suggested the possibility of translocation-mediated deregulation of the BCL2 proto-oncogene. The configuration of the BCL2 gene in MM, however, has not yet been defined using all available breakpoint probes. To address these issues, we studied 17 patients with plasma cell dyscrasias (16 MM, 1 plasmacytoma) by Southern blotting using the major breakpoint region (MBR), minor cluster region (MCR), and 5' cDNA (pB16) BCL2 breakpoint probes; with the BCL1 major translocation cluster (MTC) breakpoint probe; and with a probe to the MYC-associated MLVI-4 region (PA1.3SB). In all 17 cases, rearrangement of one or both alleles of the immunoglobulin heavy chain gene had been demonstrated, thereby confirming the presence of tumor DNA in the samples studied. None of the cases tested showed a rearrangement with the MBR BCL2 (0/16), MCR BCL2 (0/17), 5' cDNA BCL2 (0/16), BCL1 MTC (0/15), or MLVI-4 (0/15) probes. These results suggest that if BCL2 deregulation does indeed occur in MM, a mechanism other than translocation must be involved in most cases. Furthermore, rearrangements distal to the MYC gene, in the region of the MLVI-4 probe, may be less common than previously thought. Finally, a significant proportion of translocation breakpoints in band 11q13 may not be detected by the BCL1 MTC probe in MM, as is true in lymphomas.


Vimentin metaplasia in renal cortical tubules of preneoplastic, neoplastic, aging, and regenerative lesions of rats and humans.

Vimentin expression was studied immunohistochemically in renal cortical tubules of untreated male rats of various ages, rats exposed to toxins (barbital sodium, folic acid) and carcinogens (streptozotocin, N-bis(2-hydroxypropyl)nitrosamine, barbital sodium, and in humans of various ages with or without renal epithelial tumors. Fetal, neonatal, and young adult rats did not express vimentin in renal cortical tubules. Regenerative renal tubular lesions from rats with aging nephropathy and from rats with toxic nephropathy both expressed vimentin. Mitogenic lesions induced by folic acid at 24 hours, however, were not immunoreactive for vimentin. Carcinogen-induced preneoplastic renal cortical tubular lesions in rats were most often focally immunoreactive whereas strong vimentin expression was found in almost all induced renal tumors. In kidneys of three children (younger than 2 years of age), vimentin was not found in renal cortical tubular cells except in rare individual cells in one case. Vimentin was abundant in basophilic regenerative tubules in kidneys of aged individuals, however. Most (7/10) human renal carcinomas and latent preneoplastic or neoplastic renal tubular lesions found incidentally at autopsy (2/4) showed vimentin expression. The authors suggest that the switching to vimentin expression in phenotypically normal renal cortical tubular cells in rats and humans, which do not usually express the intermediate filament protein vimentin, should be considered vimentin metaplasia. Vimentin expression is dissociated from increased cell proliferation in hyperplastic and neoplastic lesions, however. Instead the degree of dedifferentiation of the tubule cells and changes in phenotype were associated with vimentin expression.


Expression of macrophage inflammatory protein-2 and KC mRNA in pulmonary inflammation.

This study sought to test the hypothesis that expression of mRNA for two cytokines, macrophage inflammatory protein-2 (MIP-2) and the KC gene product, is induced in rat lung cells during inflammatory responses in vitro and in vivo. Macrophage inflammatory protein-2 and KC are members of the platelet-factor 4 (PF-4) cytokine superfamily that cause marked neutrophil chemotaxis and activation in vitro. To investigate expression of the genes for MIP-2 and KC in rat models of lung injury, cDNA probes for these cytokines in the rat were made from polymerase chain reaction (PCR) products generated using mouse sequence-derived primers. Sequence analysis of these cDNAs showed marked homology to known murine sequences (89% and 92% MIP-2 and KC, respectively). These cDNAs were first used to study the expression of these two genes in rat alveolar macrophages (AMs) in vitro by Northern blot hybridization. Lipopolysaccharide (LPS) treatment of rat AMs in vitro caused marked increases in mRNA for both KC and MIP-2 within 30 minutes, which persisted through the 6 hours measured. To study expression during inflammation in vivo, rats were treated with LPS by intratracheal instillation. Bronchoalveolar lavage (BAL) cells and whole trachea homogenates were analyzed. There was a marked and rapid increase in MIP-2 and KC mRNA levels within both BAL cells and trachea homogenates after LPS instillation. The results support the hypothesis that MIP-2 and KC cytokines contribute to neutrophil chemotaxis and activation in this rat model of acute pulmonary inflammation.


Neuronal injury after photoactivation of photofrin II.

Photodynamic therapy has been used in the management of patients with malignant brain tumors even though the effects of this form of treatment on the adjacent normal brain are incompletely characterized. The authors examined, in sequential experiments, morphologic alterations affecting the cerebral cortex in rats injected with Photophrin II and exposed to light. Initially, minimal cell alterations, including cisternal swelling of both endoplasmic reticulum and Golgi apparatus, involved only neurons located in the superficial layers of the cerebral cortex exposed to light. These changes spread, over a period of several hours, from the surface to the bottom of the cortex and eventually involved the entire cortical segment exposed to light. The earliest structural signs of lethal injury to neurons developed over a period of 18 hours after porphyrins had been photoactivated and astrocytes had been severely damaged. Signs of lethal injury to neurons included an increase in the number of mitochondrial cristae and appearance of amorphous electron-dense deposits within swollen mitochondria. The appearance of these alterations was followed by segregation of intracytoplasmic organelles and fragmentation of nuclear and cytoplasmic membranes. The tissue changes, including those involving neurons, eventually progressed to coagulation necrosis at 48 hours. These observations suggest that prophyrins injected to rats (48 hours before photoactivation) cause swelling and necrosis of astrocytes. This is followed by neuronal necrosis, which appears at two time intervals; the initial neuronal necrosis occurs after the astrocytic disintegration. A second type of neuronal alteration appears after microvessels become thrombosed and ischemia is likely to develop.


Hyaluronan synthesis by in vitro cultured endolymphatic sac cells.

The endolymphatic sac (ELS) has been the subject of investigation for many years and yet its overall function remains unclear. It is believed mainly to be involved in the regulation of endolymph through fluid resorption and secretion of osmotically active substances. The present study was performed using in vitro cultured, fetal ELSs of 18 to 19 day gestational mice, to assess whether the ELS cells can synthesize the osmotically active polysaccharide, hyaluronan (HA). The ELS and portions of the membranous labyrinth were dissected from whole otocyst specimens and placed in 14C glucose-enhanced tissue culture media. A light microscopic (LM), autoradiographic study was performed to assess whether 14C glucose could be incorporated by the tissue into HA. Both the ELS cells and the adjacent cartilage demonstrated radiolabel incorporation within 4 hours of incubation in tissue culture medium, with increased radiolabel density in ELS cells after 24 hours of incubation. HA-specific hyaluronidase (HAase) resulted in removal of HAase-sensitive compounds in the ELS in both 4-hour and 24-hour cultured specimens when compared to adjacent cartilage cells (p = 0.001). Approximately 43 percent of the radiolabel was incorporated into HA in ELS specimens, as compared to a 22 percent HA synthesis in the adjacent cartilage tissue, suggesting preferential synthesis by ELS cells. The dissected murine otocysts demonstrate viability in vitro as measured by their ability to incorporate 14C glucose from tissue culture medium. Under these conditions the cultured ELS demonstrates an ability to synthesize HA. A theory of ELS function is proposed.


Sensory aids in conjunction with cochlear implants.

The use of cochlear implants in profoundly hearing-impaired individuals can restore varying degrees of auditory capabilities. Although very little auditory information is transmitted to these patients through amplification systems, we hypothesized that some of the cues obtained from sensory aids might be helpful when used in conjunction with the cochlear implant postoperatively. Eight patients implanted at NYU Medical Center, Bellevue Hospital Center, have used some sensory aid in addition to the Nucleus multichannel cochlear prosthesis. All subjects were evaluated using standard auditory tests including pure-tone and speech audiometry, portions of the Minimal Auditory Capabilities (MAC), Speech Pattern Contrast Perception (SPAC), Iowa test batteries; and the Early Speech Perception (ESP), Word Intelligibility by Picture Identification (WIPI), and Glendonald Auditory Screening Procedure (GASP), where appropriate. Subjects were evaluated under three conditions: implant alone, implant plus sensory aid, and sensory aid alone. Results indicate that the interaction between a multichannel cochlear implant and a sensory aid can provide some improved performance for adult implanted patients (i.e., they do better under the dual condition than in the implant-alone condition). Variables such as thresholds in the nonimplanted ear and usage time can affect the outcome.


Neural presbycusis: a diagnostic dilemma.

The combined term, sensorineural, is useful because clinical distinction between sensory and neural lesions is often difficult, and because sensory lesions may lead to secondary neural degeneration or, though rarely, a neural lesion may lead to secondary sensory degeneration. The threat of lawsuits for failure to identify treatable neurologic conditions has led to overuse of tests, while fiscal constraints exert pressure to limit expensive diagnostic testing. The purpose of this review of the relation between sensory and neural hearing loss is to provide a practical method to screen for neural lesions using pure-tone thresholds and a single speech discrimination score. The difference between the articulation index and the word recognition score of a patient provides a statistically reliable index of suspicion that may reduce the diagnostic dilemma of neural presbycusis.


Unilateral acoustic neuromas in childhood without evidence of neurofibromatosis: case report and review of the literature.

A six-year-old female with a unilateral benign acoustic neuroma and no evidence of neurofibromatosis is presented. Review of the literature revealed only 16 cases of unilateral acoustic neuromas in children less than 15 years old. Children often do not appreciate hearing loss when compared with adults. As a result, they often present with larger tumors, thus adversely affecting outcome. The need for early diagnostic tests and a hearing-conservation surgical approach is emphasized. Routine follow-up imaging studies are crucial to rule out the development of a contralateral acoustic neuroma in neurofibromatosis-2 (NF-2).


A new medication to control patulous eustachian tube disorders.

The disorder of hyperpatent eustachian tube is often unrecognized or misdiagnosed because of the ambiguous symptom of a "plugged ear." But when the patient also complains of autophony and/or hearing amphoric sounds, a patulous tube should be suspected. Persistent symptoms are distressing to most patients. This preliminary report evaluates a new nasal medication that produces closure of the tube and controls or eliminates these symptoms. It is demonstrated to be safe, predictable, and effective with little or no side effects.


Retrolabyrinthine vestibular nerve section: evaluation of technical modification in 143 cases.

Selective vestibular nerve section is effective in relieving intractable vertigo while preserving hearing. Histologic study demonstrated overlap of the cochlear and vestibular fibers at the cochleovestibular cleavage plane within the posterior fossa. In the hopes of improving surgical results by sectioning these overlapping vestibular fibers, the technique of sectioning the vestibular division of the eighth nerve was modified to include a small portion of the cochlear division. We have evaluated 143 cases of retrolabyrinthine vestibular nerve section in which this technical modification was employed. A patient questionnaire was used to examine control of vertigo as well as changes in tinnitus, unsteadiness, and disability. Hearing preservation and incidence of complications were also reviewed. Resolution or improvement of vertigo occurred in 93 percent of Menièré's cases and 74 percent of non-Menièré's cases as compared to 97 percent and 68 percent, respectively, in the pre-1986 series.


Traumatic perilymphatic fistula: how long can symptoms persist? A follow-up report.

In the past 18 years 68 ears (average 3.8 per year) were explored for perilymphatic fistula (PLF). A total of nine (13%) ears had a fistula identified at operation. Patients with a previous history of otologic surgery were excluded from this review. The most common etiology for PLF was head trauma (4 of 9). Most patients had persistent symptoms lasting months (average 6.7). Eighty-three percent of all patients had sudden or fluctuating hearing loss, 77 percent had vertigo or dysequilibrium, and 61 percent had tinnitus. Vertigo was the most commonly improved symptom postoperatively, and only 25 percent of patients had improved hearing. There were no major complications. The authors discuss indications for operation, criteria for diagnosis of PLF, and audiometric and electronystagmographic findings. This report agrees with other recent data indicating that exploration for fistula is an uncommon procedure performed by otologists.


Relevance of the Tullio phenomenon in assessing perilymphatic leak in vertiginous patients.

The Tullio phenomenon was studied in 57 control subjects, in 7 patients with confirmed PLF and in 149 patients with different types of inner ear pathology. The subjects were exposed to low-frequency sound (LFS) with sound pressure levels ranging from 130 to 132 dB and at frequencies of 25, 50, and 63 Hz. The duration of each stimulation was 30 seconds. The postural responses were measured using a force platform and analyzed with a computer. In control subjects the LFS stimulation somewhat improved the postural stability. All patients with PLF showed significantly altered postural stability during LFS exposure. Also about 20 percent of patients with labyrinthine vertigo, patients with Menièré's disease, patients with chronic middle ear disease (with vertigo) had pathologic response to LFS. None of the patients with pure sensorineural hearing loss without vertigo responded to LFS. Post-urography with LFS loading is a new, promising tool in detection of PLF, but the specificity of the test is still unsatisfactory.


Changes in hyaluronan synthesis by in vitro cultured endolymphatic sac cells.

The endolymphatic sac (ELS) has been implicated in the maintenance of endolymph volume and pressure in the membranous labyrinth through fluid resorption and secretion of osmotically active substances, known as glycosaminoglycans (GAGs). To assess whether the ELS cells synthesize the GAG, hyaluronan (HA), and to further elucidate the secretory function of the ELS, a series of experiments were carried out on in vitro tissue-cultured, fetal murine ELSs. In phase 1 of the investigation, the ELSs that were attached to a small portion of the posterior labyrinth, were resected from whole otocyst specimens and studied in tissue culture. This model was chosen to determine whether a change in endolymph homeostasis affects ELS activity. Radiolabeled 14C glucose incorporation was used to evaluate HA synthesis by ELS cells when cultured in vitro. Approximately 43 percent of the incorporated 14C glucose radiolabel was digested by Streptomyces hyaluronidase (an HA-specific hyaluronidase), suggesting HA synthesis by sac cells. In phase 2 of our experiments, the ELSs were not resected from the otocysts. Instead, they were left attached to intact membranous labyrinths, and whole otocysts were cultured. Studies analogous to those of phase 1, assessing the ability of the ELS cells to incorporate 14C glucose into HA, were performed on these specimens. Streptomyces hyaluronidase treatment of these ELS specimens resulted in a reduction in the removal of radiolabel. Therefore, the ELS cells of intact otocysts incorporated less 14C glucose into HA when compared to the ELS cells of the resected specimens.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of cochlear function in a patient with "far-advanced" otosclerosis.

A patient with "far-advanced" otosclerosis with a profound bilateral hearing loss was evaluated for cochlear reserve using transtympanic electrocochleography (ECoG) and promontory stimulation. Results indicated that both ECoG and promontory responses were present, indicating that cochlear function was present bilaterally. A stapedectomy resulted in closure of the air-bone gap. Postoperative hearing yielded excellent aided responses compared to no measurable preoperative hearing aid benefit. This case serves to demonstrate the value of evaluating cochlear function using these electrophysiologic techniques.


Facial nerve repair techniques in cerebellopontine angle tumor surgery.

This study reviewed the technique and outcome of facial nerve repair in cases of cerebellopontine angle (CPA) tumor that resulted in transection of the facial nerve. Between January 1982 and December 1989, the facial nerve was preserved in over 97.7 percent of 1110 cases of CPA tumor resection. Facial nerve repair was performed in 23 patients. The nerve repair was accomplished with either suture anastomosis or support with microfibrillar collagen. In the cases repaired with sutures, reanastomosis to a proximal facial nerve stump at the brain stem was performed with a single suture, while stabilizing the nerve with a fenestrated suction. Direct facial nerve neurorrhaphy or anastomosis with a greater auricular nerve interposition graft was successful in restoring tone and some degree of voluntary motion in 18 of the 21 patients with follow-up available (86%) and acceptable function (grade IV/VI or better) in 12 of 21 (57%). The degree of recovery of facial function was significantly better in patients without preoperative facial nerve symptoms and patients in whom the tumor did not arise from the facial nerve itself. We conclude that in cases of facial nerve transection in the CPA, good results are obtained by immediately reestablishing anatomic continuity of the nerve with direct reanastomosis or with a greater auricular nerve graft.


Extended middle fossa and retrolabyrinthine approaches in acoustic neuroma surgery: case reports.

Hearing conservation surgery for small acoustic neuromas is well accepted. At present, two approaches are primarily used: the suboccipital and the middle fossa. The middle fossa approach to the internal auditory canal has the advantage of using bony landmarks to identify and protect the facial nerve. Because of anatomic constraints presented by the superior semicircular canal however, its uses are limited to intracanalicular tumors or tumors protruding only slightly into the cerebellopontine angle. By extending the approach through the superior semicircular canal, a wide access to the cerebellopontine angle can be safely obtained. In this study three procedures, two through the superior semicircular canal and one through the posterior semicircular canal, were utilized for hearing conservation surgery. By immediately sealing off the canal ends, hearing preservation was accomplished in two out of three of these cases. The dictum that labyrinthine opening invariably leads to anacusis should be reconsidered. By utilizing approaches through the semicircular canal, it is possible that morbidity from this surgery may be reduced.


Transmission of change in the atmospheric pressure of the external ear to the perilymph.

In experiments using guinea pigs, the middle ear and perilymphatic pressures were simultaneously registered in response to pressure change in the external ear canal. In the first experiment, pressure was slowly loaded in the ear canal in the range of 200 mm H2O to -200 mm H2O. Pressure transmission to the perilymph was smaller when the bulla was open to the outside than when it was closed. It was significantly impaired by disruption of the ossicular chain and especially by closure of the round window. The data indicate that air volume in the middle ear cavity plays an important role in transmission of slowly changing atmospheric pressures. In the second experiment, the eustachian tube was closed and the pressure was changed in the range of 1000 mm H2O to -1000 mm H2O. The middle ear and perilymphatic pressures increased or decreased corresponding to the loading pressure in the range of 400 mm H2O and -200 mm H2O. Beyond these levels, response rate of the middle ear pressure decreased and perilymphatic pressure declined in spite of further increase in loading pressure. The increase in pressure difference between the middle ear and the inner ear might cause disruption of the round and/or oval windows.


Meningeal carcinomatosis producing bilateral sudden hearing loss: a case report.

Meningeal carcinomatosis is characterized by diffuse infiltration of the leptomeninges by metastatic cancer in patients usually with a previous history of malignancy. Primary tumors are usually adenocarcinomas of the breast or lung, or malignant melanoma. Meningeal carcinomatosis can present with headache and/or a variety of cranial neuropathies. We report a case of meningeal carcinomatosis presenting as a complete, bilateral, sudden hearing loss without other cranial nerve findings--a previously unreported presentation. Our patient also exhibited an unusual primary tumor site (esophagus) and histopathology for meningeal carcinomatosis. The case was impressive for the subsequent abrupt onset of a series of cranial neuropathies and the rapid deterioration in the patient's condition.


Facial nerve paralysis secondary to chronic otitis media without cholesteatoma.

Numerous papers have been written on facial nerve paralysis caused by chronic suppurative otitis media. However the authors found none documenting the results of therapy in a series of patients in whom facial nerve dysfunction was caused by chronic otitis media without cholesteatoma. Thus, there is little factual information available to help select a specific therapeutic plan for such cases. Over the past decade, the senior author has managed five cases (6 ears) of chronic suppurative otitis media without cholesteatoma in which facial paresis (4 ears) or paralysis (2 ears) developed 10 days or less before surgery. The chronic otitis media involved the mastoid and middle ear in five cases; and the mastoid, middle ear, and petrous apex in one case. Modified radical mastoidectomy was performed in four ears, tympanomastoidectomy with facial recess exposure in one ear, and complete mastoidectomy with middle cranial fossa petrous apicectomy in one ear. Five patients had complete recovery of facial nerve function (House grade I), and one patient had 90 percent recovery (House grade II). The results provide support for semi-emergent surgery in the management of chronic suppurative otitis media when facial nerve paralysis supervenes.


Comparison of apical and basal surfaces of confluent endothelial cells: patch-clamp and viral studies.

The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.


Volume-sensitive Ca influx and release from intracellular pools in gastric parietal cells.

The effects of osmotically induced changes in cell volume on cytoplasmic free Ca (Cai) were studied in parietal cells from intact rabbit gastric glands using digital image processing of fura-2 fluorescence. In resting unstimulated cells, Cai was unaffected by either cell swelling or shrinking when osmolarity was varied between 200 and 400 mosM (isotonicity 290 mosM). However, when cells were swelled in a 165 mosM solution (55% tonicity), a biphasic Ca increased was observed. On average, Cai increased transiently from 80 to 218 nM before stabilizing at approximately 140 nM. The peak was due to release from intracellular pools because it was present in Ca-free solutions while the sustained elevation was dependent on external Ca. In carbachol-stimulated cells, Ca influx was most sensitive to cell shrinkage. For example, addition of 25 mM sucrose (108% tonicity) caused a 30% decrease in the sustained carbachol-stimulated Cai increase (plateau). In contrast, carbachol-stimulated cells were relatively insensitive to cell swelling, with a 30% decrease in tonicity causing only a 15% increase in the plateau. However, as in the unstimulated cells, extreme (55% tonicity) swelling caused additional increases in Cai levels. The carbachol-dependent effects of changes in cell volume on Cai could be mimicked by treating cells with thapsigargin, an inhibitor of Ca pumps of intracellular membranes that also has been shown to stimulate Ca entry. Thus, although extreme swelling conditions (55% tonicity) could elicit Cai increases in either the presence or absence of agonist, agonist was required to observe Cai decreases due to cell shrinkage.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of oxygen consumption in fast- and slow-twitch muscle.

Phosphorus nuclear magnetic resonance spectra and steady-state O2 consumption rates were obtained from ex vivo arterially perfused cat biceps brachii (fast twitch) and soleus (slow twitch) muscles during and after periods of isometric twitch stimulation at 30 degrees C. In the biceps muscles, steady-state O2 consumption increased and phosphocreatine (PCr) concentration decreased progressively with stimulation. PCr recovery after these stimulation periods followed first-order kinetics with a half time of 10 min. The results in the biceps could be explained by a feedback control of cellular respiration by ADP concentration. In the soleus, steady-state O2 consumption also increased and PCr concentration decreased as stimulation rates increased. The half time for PCr recovery in the soleus was approximately 5 min, but, in contrast to the pattern in the biceps, the kinetics was not first order. There was an overshoot during the recovery period in the PCr content of soleus and a corresponding undershoot of Pi compared with resting values. Mitochondrial regulation by ADP is not sufficient to account for respiratory control in slow-twitch soleus. The respiration rate in neither muscle was dependent on the Pi content. Thus we conclude that the mechanism of control of cellular respiration is both quantitatively and qualitatively different in fast- and slow-twitch skeletal muscle.


Evidence for the involvement of a K+ channel in isosmotic cell shrinking in vestibular dark cells.

Cell volume changes were measured in dark cells. Isosmotic addition of 21.4 mM K+, Rb+, Cs+, or NH4+ to a control solution containing 3.6 mM K+ caused piretanide-sensitive cell swelling (initial rate for K+, 0.100 +/- 0.005 microns/s; n = 119), suggesting dependence on the Na(+)-Cl(-)-K+ cotransporter. Subsequent isosmotic removal of 21.4 mM K+ caused piretanide-insensitive cell shrinking (initial rate, -0.104 +/- 0.005 microns/s; n = 119), which was inhibited by barium, lidocaine, quinidine, quinine, verapamil, and 4-aminopyridine but not tetraethylammonium (TEA) or glibenclamide, suggesting the involvement of K+ channel(s). Barium, lidocaine, quinine, quinidine, and 4-aminopyridine caused cell swelling in control solution (initial rate for barium, 0.011 +/- 0.004 microns/s; n = 6), suggesting that the K+ channel is also involved in efflux under control conditions. Cell shrinking was slowed by 21.4 mM extracellular K+, Rb+, or Cs+ but unaffected by Na+, Li+, TEA+, or NH4+ (all in the presence of piretanide and compared with N-methyl-D-glucamine), supporting the notion that the efflux mechanism is permeable to and/or inhibited by K+, Rb+, and Cs+. Cell shrinking was slowed by the presumed replacement of intracellular K+ by Cs+ but not by Rb+. Circumstantial evidence suggests that this putative K+ channel is present in the basolateral membrane. The physiological relevance of such a K+ channel might encompass regulatory volume decrease during K+ secretion.


Ca2+ influx via Na(+)-Ca2+ exchange in immortalized aortic myocytes. I. Dependence on [Na+]i and inhibition by external Na+.

We have found that immortalized aortic myocytes have high Na(+)-Ca2+ exchange activity that is similar in amount to that of low-passage myocyte cultures. Replacing all external Na+ with K+ slightly increased 45Ca2+ uptake in cells with basal Na+ and greatly increased 45Ca2+ uptake in cells with increased intracellular Na+. External Na+ competitively inhibited 45Ca2+ uptake (inhibition constant approximately 10 mM). The dependence of exchange activity on intracellular Na+ was sigmoidal. The 50% maximum concentration (K0.5) for Na+ was 33 +/- 1 mmol/l cell water space. The Hill coefficient was 2.9 +/- 0.2, which is consistent with a stoichiometry of 3 Na+/1 Ca2+. Cytosolic free Na+ was estimated from the ratio of sodium-binding benzofuran isophthalate (SBFI) fluorescence and an in vivo calibration curve. Sulfinpyrazone, an inhibitor of anion transport, markedly increased the SBFI content of the cells without affecting cell Na+. Cytosolic free Na+ was 9 mM under basal conditions. Because free Na+ did not differ significantly from total Na+, little or no Na+ is bound or compartmentalized in these cells. The complete replacement of extracellular Na+ with K+ fails to evoke appreciable Ca2+ influx, at least in part, because of the low cell Na+ concentration relative to the K0.5 of the exchanger and its sigmoid dependence on Na+.


Ca2+ influx via Na(+)-Ca2+ exchange in immortalized aortic myocytes. II. Feedback inhibition by [Ca2+]i.

Depolarization with 50 mM K+ evoked a spike in cytosolic free Ca2+ ([Ca2+]i) and increased 45Ca2+ uptake in immortalized aortic myocytes. The following evidence indicates that the electrogenic Na(+)-Ca2+ exchanger caused the Ca2+ influx that was evoked by K+ depolarization. First, K+ depolarization had no effect on [Ca2+]i and 45Ca2+ uptake in cells with basal Na+ but strikingly increased both in Na(+)-loaded cells. Second, the [Ca2+]i increases produced by K+ depolarization depended hyperbolically on external Ca2+ (50% maximum concentration = 1.5 mM). Third, the increases in [Ca2+]i and 45Ca2+ uptake were greater when external Na+ was replaced with K+ rather than with N-methyl-D-glucamine or choline. A series of K+ depolarizations elicited a sequence of [Ca2+]i spikes, provided there was a short incubation at 5 mM K+ between the depolarizations. A prior K+ depolarization almost abolished the 45Ca2+ uptake response to K+ depolarization. The inhibition of exchange activity by a prior K+ depolarization required external Ca2+ and was completely reversible. A prior incubation with angiotensin II, platelet-derived growth factor, or ionomycin also inhibited exchange activity. Moderate [Ca2+]i increases probably feedback inhibit Ca2+ influx via the exchanger by a kinetic mechanism. Inactivation of the exchanger, together with Ca2+ extrusion or sequestration, causes the rapid decrease in [Ca2+]i from the peak evoked by depolarization.


Contractile arrest accelerates myosin heavy chain degradation in neonatal rat heart cells.

Mechanical forces influence the growth and metabolism of a variety of cells, including cultured neonatal rat ventricular myocytes. To determine whether mechanical activity affected the synthesis and turnover of myosin heavy chain (MHC) in these striated muscle cells, MHC fractional degradative rates were measured in spontaneously beating cells and in arrested myocytes in which contractile activity was prevented by L-channel blockade (with verapamil, nifedipine, nisoldipine, and diltiazem) or K+ depolarization. MHC degradative rates were measured as the difference between rates of MHC synthesis and accumulation and in pulse-chase biosynthetic labeling experiments. Both methods indicated that contractile arrest markedly increased MHC degradation. Contractile arrest produced by L-channel blockade accelerated MHC degradation to a greater extent than K+ depolarization. The signal transduction pathway linking contractile activity to alterations in MHC degradation did not involve protein kinase C (PKC), because MHC degradation was unaffected by activating PKC in arrested cells or inhibiting PKC in spontaneously beating cells. Chloroquine and E-64 did not suppress the accelerated MHC degradation, suggesting that the rate-limiting step in MHC turnover occurred before degradative processing by cellular proteinases. Using a computer simulation, we hypothesize that the rate-limiting step in MHC turnover preceded (or was coincident with) MHC release from thick filaments. Thus mechanical forces may influence MHC half-life by regulating the rate of myosin disassembly.


Effects of fat availability on acetyl-CoA and acetylcarnitine metabolism in rat skeletal muscle.

This study was designed to examine the effects of stimulation and fat availability on the contents of acetyl coenzyme A (acetyl-CoA), free CoA (CoASH), acetylcarnitine, and free carnitine in the oxidative fiber types of rat skeletal muscle. Hindlimb muscles were perfused with no exogenous free fatty acids (FFA) or high FFA (0.93 +/- 0.03 mM) for 10 min at rest and during isometric, tetanic stimulation. Soleus (SOL) and red gastrocnemius (RG) muscles were sampled prior to perfusion and following rest perfusion and 1 and 5 min of stimulation. The SOL muscle contains predominantly slow oxidative (SO) fibers and the RG contains 56% fast oxidative-glycolytic (FOG) and 35% SO fibers. O2 uptake and tetanic tension production were similar in the fat-free and high FFA treatments. Rest perfusion with high FFA increased acetyl-CoA from 14.6 +/- 1.0 to 20.1 +/- 2.5 nmol/g dry muscle (dm) and acetylcarnitine from 0.12 +/- 0.01 to 0.78 +/- 0.18 mumol/g dm in the RG, while fat-free perfusion had no effect. The SOL results were similar as high FFA increased acetyl-CoA from 7.7 +/- 1.0 to 14.2 +/- 3.1 nmol/g dm and acetylcarnitine from 0.14 +/- 0.02 to 0.49 +/- 0.09 mumol/g dm. Stimulation increased acetyl-CoA and acetylcarnitine to values above rest in SOL and RG in both treatments and removed all fat-free and high-fat differences. The decreases in CoASH and free carnitine were reciprocal to the increases in acetyl-CoA and acetylcarnitine at all time points in both muscles such that total CoA and carnitine were constant.(ABSTRACT TRUNCATED AT 250 WORDS)


Volume-sensitive basolateral K+ channels in HT-29/B6 cells: block by lidocaine, quinidine, NPPB, and Ba2+.

Volume-sensitive basolateral K+ channels were studied in apically amphotericin B-permeabilized HT-29/B6 monolayers in Ussing chambers with current fluctuation analysis. The basolateral K+ conductance and Lorentzian K+ channel noise were osmotically activated in presence of Cl- concentrations greater than or equal to 74 mM. Under isotonic conditions with 148 mM Cl-, a large transepithelial K+ current of 500 +/- 16.8 microA/cm2 and a spontaneous Lorentzian K+ channel noise with a corner frequency of 29.8 +/- 1.6 Hz (n = 31) were observed. Increasing extracellular osmolalities by addition of sucrose sensitively decreased the K+ current across the basolateral membrane. Half-maximal sucrose concentration was 20 +/- 6 mM for this shrinkage maneuver. The osmotically sensitive K+ pathway was similarly activated with the halide Br- and selective for K+ over Rb+ (4:1). The established K+ channel blockers lidocaine [50% inhibitory concentration (IC50) = 49.0 +/- 3.7 microM], quinidine (IC50 = 10.1 +/- 1.3 microM), and also the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC50 = 114 +/- 2.1 microM) completely inhibited basolateral K+ currents, whereas 46% of K+ current was blocked by barium (IC50 = 95.3 +/- 23.2 microM). Osmotic sensitivity of this K+ conductance made a correction for hypertonic effects of added blockers necessary, and considerable osmotic effects of blockers at commonly used doses were shown. All blockers induced dose dependently additional Lorentzian noise, indicating a direct inhibitory action on basolateral K+ channels. In this human Cl- secretory cell line, volume-sensitive K+ channels are localized only in the basolateral membrane and may modulate osmotic regulation when HT-29 cells swell.


Phospholipid metabolism and intracellular Ca2+ homeostasis in cultured rat hepatocytes intoxicated with cyanide.

The killing of cultured hepatocytes by 1 mM sodium cyanide was reduced by 100 microM chlorpromazine or cytochalasin B (25 micrograms/ml) or by lowering the pH of the culture medium to 6.0. In each case, ATP was depleted despite the decreased number of dead cells. The cell killing by cyanide was accompanied by an accelerated release of 3H-labeled arachidonate from phospholipids. Depletion of ATP by oligomycin did not accelerate phospholipid degradation or kill the hepatocytes. Chlorpromazine, cytochalasin B, and extracellular acidosis reduced the rate of phospholipid degradation in control cells as well as the increase that occurred with cyanide. The calcium ionophore A23187 increased phospholipid degradation and killed the hepatocytes. Chlorpromazine and extracellular acidosis, but not cytochalasin B, protected the cells and prevented the increased lipid degradation in response to A23187. After addition of cyanide, cytosolic free calcium ([Ca2+]i) did not change for 71 +/- 8 min, at which time it rose to a plateau of 683 +/- 210 nM within 10 min. A second and larger rise occurred after 84 +/- 8 min and before the death of the cells at 89 +/- 8 min. Treatment with 3.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, as well as removal of extracellular calcium, prevented these late increases in [Ca2+]i without affecting the loss of viability. It is concluded that cyanide kills cultured hepatocytes by a mechanisms that is likely related to an accelerated degradation of phospholipids. This change in lipid metabolism is not mediated by a rise in [Ca2+]i but rather may relate to an alteration in the interaction between the cytoskeleton and the plasma membrane.


Ionic basis for spontaneous depolarizations in isolated smooth muscle cells of canine colon.

The type of cell that serves as the pacemaker in the colon is presently unknown. This study evaluated the ionic basis of spontaneous depolarizations in circular smooth muscle cells isolated from canine colon using whole cell voltage and current clamp techniques. Increasing temperature increased the probability of observing spontaneous depolarizations, depolarized the resting membrane potential (RMP), and increased Ca2+ and K+ currents. Spontaneous depolarizations occurred as rhythmic events, in bursts, or as isolated events. Varying the holding potential from -100 to -40 mV inhibited a component of inward current thought to be necessary for spontaneous depolarizations. The Ca2+ channel blockers, nickel and nisoldipine, inhibited spontaneous depolarizations. Nickel caused a hyperpolarization, whereas nisoldipine did not affect RMP. Ouabain depolarized the RMP and inhibited spontaneous depolarizations. The K+ channel blocker, tetraethylammonium, depolarized the RMP and lengthened the duration of spontaneous depolarizations. The key finding is that single colon circular smooth muscle cells are capable of generating spontaneous depolarizations similar to those described for slow waves in intact tissues and that a temperature- and nickel-sensitive inward current is essential for spontaneous activity.


A novel remote-sensing isometric force transducer for micromechanics studies.

We have developed an innovative transducer for measuring force with femtonewton-to-micronewton resolution in biological systems. A magnetic microsphere is attached to the specimen being studied and is positioned between two electromagnets. Video microscopy and edge detection are used to monitor small movements of the microsphere that occur when the specimen generates force. An automatic control system adjusts the current through the electromagnets to keep the microsphere stationary. Measured force is a linear function of this current. This transducer is unique in its combination of sensitivity and isometric properties and its ability to measure force without direct connections to the specimen. That is, the transducer is "remote sensing" and can measure force through intervening membrane or tissue. The transducer is isometric at steady state to the limit at which displacement of the microsphere can be resolved, which can be as low as 19 nm. The completed system is being used to study the mechanics of interstitial connective tissue but may also be used to study molecular generation of force.


Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle.

It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.


Effects of thyroid hormones on urinary and renal kallikreins.

The effects of thyroid hormones on the urinary excretion of kallikrein and on renal kallikrein were studied in rats. Total and active urinary kallikrein was decreased after thyroidectomy, but renal kallikrein content remained unchanged. Diuresis increased, and kidney weight and plasma aldosterone concentration decreased. Treatment with 3,5,3'-triiodo-L-thyronine restored the urinary kallikrein in thyroidectomized rats to normal and increased it in intact rats. It also produced increases in kidney weight and plasma aldosterone and a decrease in diuresis. The effect of thyroid hormones on the urinary kallikrein response to mineralocorticoids was also tested. Deoxycorticosterone acetate increased urinary kallikrein more in normal than in thyroidectomized rats. These results suggest that thyroidectomy decreases renal kallikrein synthesis and lowers the turnover rate of the enzyme, changes not detectable by a single measurement of the renal kallikrein content but reflected by an alteration in the urinary excretion of the enzyme. Thyroid hormones participate in the control of urinary kallikrein. This effect, however, is probably indirect and may be mediated by mineralocorticoids since thyroid function affects both the plasma level of aldosterone, which is known to influence renal kallikrein, and the kallikrein response to exogenous mineralocorticoids.


Plasma GSH/GSSG affects glucose homeostasis in healthy subjects and non-insulin-dependent diabetics.

In healthy subjects (n = 10) and non-insulin-dependent (type II) diabetics (n = 10) matched for age [43.1 +/- 2.2 vs. 41 +/- 4.4 yr, P = not significant (NS)], body mass index (25.1 +/- 1.1 vs. 26 +/- 0.8 kg/m2, P = NS), gender ratio [5 males (M)/5 females (F) vs. 5M/5F], and mean arterial blood pressure (105 +/- 7 vs. 106 +/- 9 mmHg, P = NS), we determined the changes in insulin secretion and action after glutathione infusion (15 mg/min) and the relative increase in the plasma reduced (GSH)/oxidized (GSSG) glutathione ratio. The rise in the plasma GSH/GSSG ratio significantly improved total body glucose disposal in healthy subjects and in diabetic patients. In this latter group, GSH infusion potentiated the beta-cell response to glucose slightly. In controls and diabetics, insulin infusion with a simultaneous increase in the plasma GSH/GSSG ratio significantly enhanced nonoxidative glucose disposal without affecting oxidative glucose metabolism. After glutathione infusion, all metabolic and hormonal changes correlated with a significant decline in plasma membrane microviscosity. In conclusion, the plasma GSH/GSSG ratio seems to play a major role in the modulation of glucose homeostasis mainly in diabetics.


Thermogenesis after surgery: effect of perioperative heat conservation and epidural anesthesia.

Body temperature, respiratory gas exchange, and plasma catecholamines were determined before and after surgery in three groups [control (C), warmed (W), and epidural (E) who received local anesthetic at T4-S5 dermatomes during and for 24 h after surgery] of patients undergoing colonic surgery under general anesthesia. At the end of surgery, group W were nursed in an ambient temperature of 28-30 degrees C, whereas the others were at 20-23 degrees C for a period of 24 h. Core (Tc) and dorsal hand temperature decreased during surgery in both C and E (P less than 0.05) but not in W. After surgery, Tc increased similarly in C and E and by a smaller amount in W. Plasma catecholamine concentrations increased significantly in C and W but not in E (P less than 0.001), with the greatest response occurring in C. Postoperative oxygen consumption and carbon dioxide production exceeded preoperative values (P less than 0.01) in C but not in W or E. After surgery, plasma albumin fell and C-reactive protein increased similarly in all three groups. Thus body heat conservation or epidural blockade attenuates or abolishes the rise in plasma catecholamines and oxygen consumption postoperatively but does not prevent the increase in Tc or the acute phase protein response.


Energy metabolism and aging: a lifelong study of Fischer 344 rats.

Metabolic rate is widely regarded as an important component of aging processes, but variation of metabolic rate with age has not been well characterized. The purpose of the present study was to measure daily metabolic rate under usual living conditions over the lifespan of barrier-maintained Fischer 344 rats. In addition, effects of life-prolonging food restriction were assessed. Metabolic rate was measured indirectly by analysis of gas entering and leaving standard rodent cages over a 24-h period. Group A rats were fed ad libitum. Group B rats were fed 60% of ad libitum intake from 6 wk of age. Both group A and group B rats exhibited variation of metabolic rate per unit lean mass over the lifespan, with metabolic rate decreasing from 6 to 18 mo and then increasing from 18 to 24 mo of age. Results show estimates of lifetime energy expenditure in rats should take account of variability of metabolic rate and confirm the life-prolonging action of food restriction is not a consequence of reduced metabolic rate per unit metabolic mass. Rather, restricted rats are able to sustain appropriate fluxes of nutrients and appropriate metabolic rate under conditions of fuel utilization which promote maintenance of cellular homeostasis.


Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability.

Incorporation of L-[1-13C]leucine into muscle protein and leg exchange of L-[15N]phenylalanine were used to assess the effects over 240 min of amino acid supply on leg protein turnover in anesthetized, overnight-fasted (Landrace x Great White) female pigs. In all pigs, plasma insulin and glucagon stability was ensured by infusion of somatostatin (8 micrograms.kg-1.h-1), insulin (6 mU.kg-1.h-1), and glucagon (72 ng.kg-1.h-1). Mixed amino acid infusion (260 mg.kg-1.h-1) caused a 2- to 2.5-fold elevation of arterial plasma phenylalanine and leucine; in a control group (no amino acid infusion), an increase in phenylalanine and leucine concentration was observed as a result of the hormone clamp. Plasma insulin and glucagon concentrations were steady and not significantly different between control and amino acid-infused groups during the final 240 min, but plasma glucose fell (P less than 0.05) in both groups (4.57 +/- 0.17 to 3.15 +/- 0.73 mM). Muscle protein synthetic rate (estimated from the change in L-[1-13C]leucine incorporation compared with labeling of [13C]leucyl-tRNA) was greater in amino acid-infused (0.076%/h) than in control (0.053%/h) pigs. In the control group, leg amino acid balance was negative (Phe alone, -10.2 +/- 9.4 nmol Phe.100 g-1.min-1; total amino acids, -0.27 +/- 1.04 micrograms amino N.100 g-1.min-1), but during amino acid infusion, balance was positive (Phe alone, +33.6 +/- 8.8 nmol Phe.100 g-1.min-1; total amino acids, +58.2 +/- 4.9 micrograms amino N.100 g-1.min-1).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of long-term rhGH administration in GH-deficient adults on fat cell epinephrine response.

Besides exerting its own lipolytic effect, growth hormone (GH) has been reported to potentiate the lipolytic response of adipose tissue to epinephrine. It was thought interesting to find out whether long-term recombinant human growth hormone (rhGH) administration modifies epinephrine-induced lipolysis in isolated adipocytes of GH-deficient adults. In a double-blind protocol, GH-deficient subjects received either 6 mo placebo (controls, n = 5) or 6 mo rhGH (treated, n = 5). Biopsies of fat were obtained from the periumbilical region before and after placebo or rhGH administration. The response of the collagenase-isolated fat cells to various concentrations of epinephrine was assessed by glycerol release, measured by bioluminescence. Epinephrine-induced lipolysis was not altered by 6 mo placebo, while it was significantly increased by 6 mo rhGH. A similar response was obtained with isoproterenol, but no significant differences occurred in either group with UK 14304, an alpha 2-adrenoreceptor agonist. Thus, in GH-deficient adults, long-term rhGH administration improves the lipolytic response of isolated adipocytes to epinephrine, essentially by increasing the efficiency of the beta-adrenergic pathway.


Uptake of glucose carbon in muscle glycogen and adipose tissue triglycerides in vivo in humans.

Glucose disposal into muscle glycogen has previously been directly studied after intravenous, but not after physiological, oral administration. In this study 100 g glucose, containing 50 microCi [U-14C]glucose, were taken orally by premenopausal women in the overnight fasting state or after a carbohydrate-rich meal. After 4 h, biopsies were taken from the vastus lateralis muscle and abdominal and femoral subcutaneous adipose tissue for determinations of label in isolated glycogen and triglycerides, respectively. Uptake of administered glucose carbon in muscle glycogen was estimated to be approximately 20 g in the fasting and approximately 9 g in the carbohydrate-fed groups, respectively. Total uptake in adipose tissue triglycerides was approximately 2 g. Glucose carbon uptake in muscle glycogen correlated positively with glucose infusion rates during euglycemic hyperinsulinemic glucose clamps, glycogen synthase activity in vitro, and type I muscle fiber distribution and fiber area. Negative correlations were found between glucose uptake in adipose tissue triglycerides, type II fiber distribution and fiber area, as well as the waist-to-hip circumference ratio and degree of obesity. It was concluded that muscle glycogen is probably not a major pathway for disposal of oral glucose carbon in the sedentary condition.


Dexamethasone acts locally to inhibit longitudinal bone growth in rabbits.

Excess glucocorticoid is a potent inhibitor of epiphysial growth. Several mechanisms have been suggested to explain this growth inhibition, including both direct local effects of glucocorticoid on the epiphysial growth plate and indirect systemic effects. Previous studies do not distinguish which of these proposed mechanisms is actually responsible for the growth suppression in vivo. To resolve this controversy, we developed a method for delivering glucocorticoid directly into the rabbit epiphysial growth plate and for accurately measuring the resulting epiphysial growth rate. Five-week-old male rabbits received a local infusion of dexamethasone phosphate (80 ng/microliters, 1 microliters/h) into one proximal tibial growth plate and an infusion of vehicle into the contralateral growth plate. Growth rate was determined by inserting metal pins into the bone immediately adjacent to the growth plate and measuring the change in distance between pins on serial radiographs. This method permitted growth rates to be measured over intervals as short as 3 days, with an error of approximately 5%. Local dexamethasone administration decreased proximal tibial growth rate by 77% compared with the contralateral vehicle-treated tibia (P less than 0.0001). We conclude that excess glucocorticoid causes a rapid potent inhibition of growth by a direct local action on the growth plate.


On the mechanism of stimulation of ureagenesis by gluconeogenic substrates: role of pyruvate carboxylase.

Gluconeogenic substrates, lactate or pyruvate, or ornithine produced 100% increase of urea synthesis from NH4Cl. The combined administration of ornithine and lactate (or pyruvate) produced more than additive effects, indicating that they acted at different steps in a potentiating manner. The uptake of ornithine was enhanced by gluconeogenic substrates. This finding may explain, at least in part, the stimulating effect of these substrates on ureagenesis from NH4Cl and ornithine. The gluconeogenic substrate-induced stimulation of ureagenesis from NH4Cl was still observed under conditions of reduced flux through pyruvate carboxylase, ruling out that their action was exclusively mediated by the anaplerotic effect of this enzyme. Pyruvate was a more potent stimulator of ureagenesis than lactate and its effect less sensitive to pyruvate carboxylase inhibition. These observations indicate that a correlation exists between stimulation of ureagenesis by gluconeogenic substrates and flux through pyruvate dehydrogenase. It is concluded that gluconeogenic substrates may stimulate ureagenesis from NH4Cl by 1) increasing intracellular ornithine availability and/or 2) enhancing flux through pyruvate dehydrogenase and consequently the tricarboxylic acid cycle activity.


Regulation of expression of the lipoprotein lipase gene in brown adipose tissue.

The regulation of lipoprotein lipase gene expression in brown adipose tissue was studied. Rats were preacclimated to 21 degrees C. Exposure to cold (4 degrees C) resulted in a rapid increase in the level of lipoprotein lipase mRNA in the tissue. The level peaked (expressed per microgram total RNA) after approximately 8 h and then slowly declined. The increased lipoprotein lipase mRNA level was not due to an increased stability of the mRNA, but, in a transition event from a high to a low expression of the lipoprotein lipase gene, a transcription-dependent process was recruited that accelerated the breakdown of lipoprotein lipase mRNA. Norepinephrine injections increased lipoprotein lipase mRNA levels in the tissue; this effect was mediated via a beta-adrenergic receptor. The effect of cold could be mimicked by norepinephrine injections, and these two effects were not additive, indicating that the cold effect was mediated by norepinephrine. The lipoprotein lipase mRNA level was also increased by insulin injections (into fasted animals); thus an increase in lipoprotein lipase gene expression in brown adipose tissue may be induced via two different stimuli, which, intracellularly, would be mediated via different signaling systems. In all investigated conditions, the changes in lipoprotein lipase mRNA levels observed here were parallelled by alterations in lipoprotein lipase activity reported earlier from this laboratory. It was therefore concluded that, under the conditions studied, lipoprotein lipase activity in brown adipose tissue was primarily regulated at the transcriptional level.


Evidence for the modulation of cell calcium by epinephrine in fish hepatocytes.

The effect of epinephrine (10(-7) M) on cytosolic free-Ca2+ concentration ([Ca2+]i) and its dependency on external Ca2+ were studied in fura-2-loaded hepatocytes isolated from three teleost fish species: American eel, brown bullhead, and rainbow trout. Basal [Ca2+]i was similar in eel and trout hepatocytes (79.6 +/- 14.6 and 75.7 +/- 17.4 nM, respectively) but was significantly higher in bullhead cells (184 +/- 23 nM). Epinephrine-induced [Ca2+]i oscillations were observed only in eel hepatocytes. These oscillations, which presented variable patterns among individual cells, also developed in the absence of external Ca2+, although their amplitude progressively declined to eventually vanish under such conditions. In bullhead hepatocytes, epinephrine induced a biphasic [Ca2+]i response, with an initial transient rise followed by a sustained component; this response was virtually abolished in the absence of extracellular Ca2+. The peak [Ca2+]i achieved (433.5 +/- 135.6 nM) was more than two times that of eel cells (184.3 +/- 30 nM) but represented a similar percent increase above control [Ca2+]i for both species. Rainbow trout hepatocytes, contrary to eel and bullhead cells, demonstrated little epinephrine sensitivity, with less than 20% of the cells responding. These data clearly point to significant species differences both in terms of epinephrine-induced changes in [Ca2+]i and in the dependence of these transients on external Ca2+. Thus the eel response relies primarily on intracellular stores, whereas the bullhead response principally involves enhanced influx of Ca2+ from the extracellular milieu. Furthermore, the similarity of these responses with those reported for mammalian hepatocytes strongly suggests that an alpha-adrenoceptor/Ca2+ transduction system is involved in at least eel and bullhead hepatocytes.


Vasoconstrictor-induced secretion of ANP in fetal sheep.

Fetal secretion of atrial natriuretic peptide (ANP) increases during volume expansion and hypoxia. It is unknown whether this is associated with alterations in right atrial pressure (RAP) or distension and whether increases in ANP secretion reflect effects of specific vasopressors. To address this we studied fetal sheep (n = 13) at 125-140 days gestation, infusing either angiotensin II (ANG II; 0.023-5.73 micrograms/min) or the alpha-agonist phenylephrine (Phen; 0.031-7.64 micrograms/min) while monitoring mean arterial pressure (MAP), RAP, heart rate, and amniotic sac pressure. Arterial blood was obtained before and at 5 min of infusion to measure ANP, blood gases, and pH; umbilical venous blood was collected to determine placental clearance of ANP. ANG II caused dose-dependent increases in MAP and plasma ANP (P less than 0.05), whereas Phen caused dose-dependent increases in MAP, but ANP rose only with the highest dose (40 +/- 12%). delta MAP and delta RAP were highly correlated for Phen (r = 0.74, P = 0.002) and ANG II (r = 0.90, P less than 0.001), but for both agents the increase in delta RAP was proportionately greater than delta MAP, and increases in plasma ANP were greater per millimeter mercury rise in RAP than that observed with MAP. Increases in ANP were associated with a dose-dependent rise in hematocrit, suggesting decreases in intravascular volume. There was no fetal placental clearance of ANP. As in adults, ANG II- and alpha-agonist-induced fetal ANP secretion appears to primarily reflect increases in RAP and thus right atrial distension.


Thyroid hormone effects on cardiac gene expression independent of cardiac growth and protein synthesis.

Prior studies have demonstrated the importance of hemodynamic loading in mediating thyroxine (T4)-induced cardiac hypertrophy. Direct cellular effects of thyroid hormone have been implicated in modulating the expression of the myosin heavy chain (MHC) genes and the slow sarcoplasmic reticulum calcium adenosine triphosphatase (SR Ca(2+)-ATPase) gene. In the present report, administration of T4 for 72 h did not stimulate growth of the hemodynamically unloaded heterotopic isograft. The synthetic rates of total cardiac proteins and MHC in the isograft remained significantly lower at 64 and 53% of the respective rates measured simultaneously in the in situ working heart. Although total left ventricle RNA content in the isograft was unchanged by T4, alpha-MHC and SR Ca(2+)-ATPase mRNA concentrations were increased 181 and 208%, respectively, and the previously observed beta-MHC expression was completely prevented. These data indicate that, although T4 requires an increased hemodynamic load to stimulate cardiac protein synthesis, it is capable of directly altering the expression of at least two myocyte-specific genes. Therefore some of the phenotypic alterations observed with thyroid hormone treatment are the result of direct effects of the hormones on specific cardiac genes and independent of changes in cardiac growth.


Importance of basal glucagon in maintaining hepatic glucose production during a prolonged fast in conscious dogs.

We undertook studies in conscious dogs to assess the role of basal glucagon in stimulating glucose production after a 7-day fast. Two protocols consisting of a 40-min basal period (-40 to 0 min), and a 180-min test period (0-180 min) were used. During the test period of the first protocol (hormone replacement; n = 4), somatostatin was infused (0.8 micrograms.kg-1.min-1) along with basal intraportal replacement amounts of insulin and glucagon, whereas in the second protocol (glucagon deficiency; n = 5), somatostatin plus insulin alone were infused. Glucose production and gluconeogenesis were measured using tracer and arteriovenous difference techniques. Plasma insulin levels were similar during the test period in both protocols (6 +/- 1 microU/ml). The plasma immunoreactive glucagon level in the control protocol averaged 50 +/- 8 pg/ml, whereas in the glucagon-deficiency protocol the level fell from 50 +/- 8 to 29 +/- 8 pg/ml (P less than 0.05). The plasma glucose level and the rate of glucose production were unchanged during bihormonal replacement. During glucagon deficiency the plasma glucose level was held constant at 100 +/- 4 mg/dl by glucose infusion. Tracer-determined endogenous glucose production fell from 1.8 +/- 0.1 to 1.0 +/- 0.1 mg.kg-1.min-1 by 30 min (P less than 0.05). After 3 h of glucagon deficiency, gluconeogenic conversion of alanine to glucagon was reduced 40% and the hepatic fractional extraction of alanine was reduced by 45%. The efficiency of the gluconeogenic process within the liver was not altered by glucagon deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)


Interactions between insulin and glucocorticoids in the maintenance of genetic obesity.

Glucoregulation and body composition were examined in 3-mo-old C57BL/6 ob/ob mice 6 wk after streptozotocin (STZ) or STZ plus adrenalectomy. STZ depressed somatic growth in ob/ob mice but did not cause hyperglycemia until immunoreactive insulin (IRI) was 40% (100 microU/ml) that of intact ob/ob mice. When IRI approached that of lean mice (40 microU/ml), ob/ob mice displayed severe hyperglycemia (800+ mg/dl) and other sequelae of type I diabetes but still maintained the same 50% body fat as untreated obese mice. In contrast, STZ diabetes in lean mice caused disproportionate reductions in body fat. Adrenalectomy before STZ led to the same insulinopenia, depressed growth, and hyperglycemia as STZ alone, but, after combined treatment, percent body fat declined in proportion to IRI. Thus a subgroup of severely diabetic adrenalectomized STZ obese mice with very low IRI (20 microU/ml) had body fat contents and fat-free masses equal to those of weight-matched lean mice. The data suggest that hypercorticoidism rather than hyperinsulinemia is largely responsible for obesity in ob/ob mice. However, in the absence of adrenal glucocorticoids, or perhaps with just their normalization, hyperinsulinemia appears necessary for maintaining excessive body energy stores.


Fasting and lactate unmask insulin responsiveness in the isolated working rat heart.

We have previously reported that the nutritional state in vivo results in differential insulin responses by the perfused heart in vitro. To further assess the effects of insulin on glucose uptake at physiological work loads, hearts from fed and fasted (16-20 h) rats were perfused with buffer containing 2-[18F]fluoro-2-deoxy-D-glucose (2-FDG) and glucose (10 mM) alone or plus lactate (10 mM) as a competing substrate, with insulin (10 mU/ml) added after a control period. When glucose was the only substrate, the addition of insulin decreased the fractional rate of 2-FDG uptake in hearts from either fed or fasted rats. The effect of insulin on increasing myocardial 2-FDG uptake was immediate and sustained only in hearts from fasted rats in the presence of lactate, despite no change in cardiac work. At the same time, the increase in 2-FDG uptake and phosphorylation was associated with an increase in the tissue content of glycogen in hearts from fasted rats. We conclude that lactate unmasks insulin sensitivity in heart muscle at physiological work loads but that this unmasking of insulin-mediated glucose uptake is dependent on the nutritional state of the animal. The glucose up as a result of insulin stimulation is preferentially utilized for glycogen repletion and does not enter the glycolytic pathway. This observation also suggests that myocardial glycogen synthesis in vitro is affected by the nutritional state in vivo and that lactate provides a substrate for oxidative phosphorylation while glucose is preferentially utilized for glycogen synthesis.


Pretranslational regulation of two cardiac glucose transporters in rats exposed to hypobaric hypoxia.

To investigate the mechanism by which cardiac glucose utilization increases during hypoxia and increased work load, we studied the effect of 2 and 14 days of hypobaric hypoxia on the expression of two subtypes of the facilitative D-glucose transporter, the GLUT-4 or "insulin-regulatable" isoform and the GLUT-1 isoform thought to mediate basal transport. Rats lose weight when exposed to hypobaric hypoxia, so fasting controls were used in the 2-day studies and pair-fed controls in the 14-day experiments. Hypobaric hypoxia (PO2 69 mmHg) resulted in right ventricular (RV), but not left ventricular (LV), hypertrophy. RV and LV GLUT-1 mRNA levels increased 2- to 3-fold after 2 days and 1.5- to 2-fold after 14 days of hypobaric hypoxia compared with both fasted rats and normal controls. RV GLUT-1 protein increased approximately 3-fold and LV GLUT-1 protein increased 1.5-fold after 14 days of hypobaric hypoxia vs. both pair-fed and normal controls. RV GLUT-4 mRNA decreased to 26% and RV GLUT-4 protein decreased to 54% of normal control levels as a result of 2 days of hypobaric hypoxia. RV GLUT-4 mRNA decreased to 64% of normal control levels with no change in RV GLUT-4 protein as a result of 2 days of fasting. We conclude that hypobaric hypoxia increases cardiac GLUT-1 expression at the pretranslational level in both ventricles. The greater increase in GLUT-1 protein on the right suggests an additive effect of pressure overload. GLUT-4 expression is reduced early in the development of RV hypertrophy.


Plasma carnitine in fasting neonatal and adult northern elephant seals.

Maintenance of adequate body carnitine stores is a requisite for fasting mammals, whose energy is derived mainly from free fatty acid oxidation. The impact of longterm fasting on carnitine status is unclear, and there have been no reports of carnitine during naturally occurring fasts. Total (TC), free (FC), and acylated (AC) plasma carnitine levels were determined in 10 weaned and 11 adult northern elephant seals (Mirounga angustirostris) during natural fasts lasting from 1 to 3 mo. In pups, TC declined little and AC increased only slightly [P greater than 0.05, analysis of variance (ANOVA)] through 11 wk of fasting. Plasma FC dropped by 53 and 26% from week 1 values at 10 and 11 wk fasting, respectively (P = 0.014, ANOVA). The AC/FC ratio did not approach 1.0 until 7 wk of fasting. TC was 38.6 +/- 1.4 microM and 47.6 +/- 4.1 microM in adult females and males, respectively. Adult AC/FC ratios were 0.71 +/- 0.10 (females) and 0.08 +/- 0.04 (males). Plasma TC status is not negatively affected by extended fasting in adult and weaned northern elephant seals. These data support the hypothesis that fasting northern elephant seals defend plasma TC and maintain an attenuated AC/FC ratio well into their prolonged natural fast.


Single umbilical artery ligation-induced fetal growth retardation: effect on postnatal adaptation.

To assess whether prolonged intrauterine stress and resultant fetal growth retardation result in depletion of adrenal catecholamines and alter the adrenergic signal transduction system, we studied newborn sheep after single umbilical artery ligation (SUAL)-induced growth retardation. The animals were delivered at term, and postnatal cardiovascular, pulmonary, endocrine, and metabolic responses were measured. We also evaluated the status of myocardial and pulmonary beta-adrenergic receptor number and function. SUAL caused significant growth retardation but relative preservation of brain and adrenal gland weight and adrenal catecholamine content. Blood pressure, plasma free fatty acid, and glucose responses at birth were blunted in SUAL animals. The plasma epinephrine (Epi) and norepinephrine levels were comparable in both groups for the first 2 h of age. By 4 h, both plasma concentration and plasma appearance rate of Epi were reduced to 40% of control in SUAL animals (P less than 0.05). Neither beta-receptor density, affinity, nor adenylate cyclase activity were altered by SUAL in either cardiac or pulmonary membranes. These results suggest that, rather than overt depletion, there is relative sparing of initial adrenal medullary function that later waned. This response and preservation of the beta-adrenergic signal transduction system may represent partial compensation for the physiological stress induced by SUAL.


Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects.

In metabolic tracer studies it is frequently useful to infuse tracers that are differently labeled variants of the same molecule. These tracers are known as isotopomers. Analysis of the enrichment of each isotopic analogue can be accomplished by gas chromatography-mass spectrometry (GC-MS). However, the raw GC-MS data must be corrected to give the information required. This paper addresses how to transform the raw GC-MS data, consisting of relative abundance ratios at specific ion masses, into relative molar ratios of tracer and tracee molecules. Several correction factors are necessary. First, the background must be measured and corrected for, since it is always present in the sample. Second, the abundances in the spectrum of the labeled molecule are different from those in the unlabeled molecule, and this proportionality "skew" is corrected. A third correction factor accounts for the overlapping spectra of two or more isotopomers that cannot be measured independently. The final correction removes the "double vision" effect that may appear in some spectra due to the presence of (M - H)+ species.


Hypoxic liver injury and the ameliorating effects of fructose: the "glucose paradox" revisited.

It has been independently postulated that nutritional status is a modulator of the hepatic injury response to hypoxia and that glucose may be a poor substrate for hepatocellular metabolism. This study provides data linking these two concepts within the framework of metabolic zonation of the liver. With the use of a hypoxically perfused isolated rat liver model, cellular injury, as reflected by aspartate aminotransferase (AST) release, was significantly greater in the liver of fasted (mean AST 489 U/g liver at 3 h) than fed (40 U/g) animals. The extent of injury during hypoxia was decreased to a comparable degree in fasted livers perfused with Wisconsin solution (27 U/g) or 20 mM fructose (51 U/g). Perfusion with (11.5 mM) glucose plus insulin provided no hepatoprotection (791 U/g); however, supraphysiological amounts of glucose (100 mM) with (310 U/g) or without (321 U/g) insulin (10 U) or dihydroxyacetone (220 U/g) provided a modest reduction in AST release. Cellular injury measured by trypan blue uptake showed a marked zonal pattern, with upstream regions incurring greater parenchymal and nonparenchymal injury than downstream areas. These data that indicate that exogenous glucose is poorly utilized as an energy substrate by the liver during hypoxia are consistent with data from the fasted-refed rat model, suggesting a "glucose paradox" in the liver. The findings also suggest that low levels of oxygen are an important factor mediating "hypoxic" liver injury.