Ultrasound visualization of hepatic peribiliary cysts: a comparison with morphology. We report herein a hitherto unrecognized, interesting ultrasound finding ("hilar multicystic echo complex"), the result of peribiliary cysts in the liver. This ultrasound finding was discovered around intrahepatic large bile ducts and large portal vein branches near the hepatic hilum in an autopsy case with hepatocellular carcinoma, submassive hepatic necrosis superimposed on chronic active hepatitis, and portal hypertension. Antemortem ultrasound examination revealed the hilar multicystic echo complex around the portal venous branches near the hepatic hilum. Autopsy confirmed that the hilar multicystic echo complex was due to peribiliary cysts that were present around the bile ducts at the hilum. The peribiliary cysts were thought to have arisen from cystic dilatation of preexisting intrahepatic peribiliary glands. These peribiliary cysts reportedly occur in livers with portal hypertension (e.g., cirrhosis, hepatocellular carcinoma, idiopathic portal hypertension, extrahepatic portal obstruction, and portal thromboembolism), adult-type polycystic disease of the liver and kidneys, and systemic infection. Therefore, recognition of peribiliary cysts at sonography would have diagnostic value, and may indicate that presence of one of the above described liver diseases.


Extensive portal tumor thrombi with portal hypertension in an autopsy case of intrahepatic cholangiocarcinoma. Vascular invasion is not a prominent feature of cholangiocarcinoma (CCC), in contrast to hepatocellular carcinoma (HCC), which frequently shows extensive vascular tumor thrombi. We report an autopsy case of CCC with extensive portal tumor thrombi and portal hypertension. A 57-yr-old man presented with abdominal pain. Liver imaging revealed no tumors, but showed intrahepatic portal venous obstruction. HCC with portal tumor thrombi was suspected clinically. His clinical course was rapid; he died of hepatic failure 50 days after admission. At autopsy, the liver (2,700 g) was studded with diffuse whitish yellow granular areas with flecks of coalescent granules. Intrahepatic portal veins were diffusely occluded by tumor thrombi. Microscopically, the tumor was poorly differentiated adenocarcinoma with mucin; tumor cells were immunohistochemically positive for carcinoembryonic antigen, CA 19-9, DU-PAN-2, and biliary type cytokeratins, but negative for alpha-fetoprotein. Tumor cells were diffuse in the liver, and there were numerous tumor thrombi in the small portal veins. Hepatic veins and small arteries were occasionally occluded by tumor thrombi. There was ascites, splenomegaly and tumor thrombi in the gastric and esophageal veins, suggesting that portal hypertension had been present. This tumor seemed to have marked affinity to invade portal veins. It must be stressed that there are CCCs with extensive portal tumor thrombi and resultant portal hypertension.


Second primary cancers following anal and cervical carcinoma: evidence of shared etiologic factors. The authors examined the incidence of second primary cancers occurring after cervical and anal cancer. Data from the Connecticut Tumor Registry for 1935-1988 and eight other US tumor registries for 1973-1988 were used. Women with primary invasive cervical cancer had a relative risk of 4.6 (95% confidence interval (CI) 2.4-8.1) for subsequent invasive anal cancer. Increased relative risks after cervical cancer were also found for cancers of the oral cavity (relative risk (RR) = 2.2), stomach (RR = 1.5), rectum (RR = 1.4), larynx (RR = 3.4), lung (RR = 3.0), vagina (RR = 5.6), bladder (RR = 2.7), for kidney (RR = 1.9); decreased relative risks were noted for melanoma (RR = 0.5) and breast cancer (RR = 0.8). Patients with a primary diagnosis of anal cancer had relative risks for subsequent invasive and in situ cervical cancer of 1.3 (95% CI 0.2-4.5) and 3.4 (95% CI 0.9-8.8), respectively. Anal cancer was also associated with increased relative risks of subsequent lung (RR = 2.5) and prostate (RR = 1.8) cancers, whereas the relative risk of uterine cancer was 0.2 (95% CI 0.0-0.9). These findings support other evidence for common factors, such as human papillomavirus infection and cigarette smoking, in the etiology of cervical and anal cancer.


Malignant T gamma/delta lymphoproliferative disease with natural killer lytic activity. A 17-year-old girl presented with a lymphoproliferative disease involving the bone marrow, peripheral blood, and liver associated with reactive hyperplasia of the spleen. Neoplastic cells were atypical medium-sized lymphoblasts with convoluted nuclei and nucleoli without features of large granular lymphocytes (LGL). The phenotype was CD3+ CD4- CD8-, TCR alpha/beta-, TCR gamma/delta+, delta TCS1-, and CD16+, and these cells exhibited spontaneous natural killer (NK) activity. DNA analysis showed rearrangement of the TCR gamma gene but not of TCR beta or of Ig mu genes. This unusual lymphoproliferative disease may represent the neoplastic expansion of a minor subset of normal T gamma/delta cells with NK activity.


IgD myeloma presenting as a testicular tumor: establishment and characterization of an IgD-secreting myeloma cell line. A 72-year-old man presented with a left testicular tumor and underwent orchiectomy. The tumor was massively infiltrated with myeloma cells bearing monoclonal cytoplasmic IgD lambda. Three months after orchiectomy, he developed huge abdominal masses and subsequently ascites containing numerous myeloma cells. An IgD-secreting myeloma cell line, designated delta-47, was established from the ascites. This cell line expressed CD4 and CD38, but lacked Fc and complement receptors, surface immunoglobulin, CD19, HLA-DR, and PCA-1. CD30 was detected on the cultured cells but not on the ascites tumor cells. Delta-47 cells secreted the same immunoglobulin (IgD lambda) as was found in the patient's serum. The light chain had a molecular weight of 35 kD which was larger than that of the normal light chain. Chromosome analysis of delta-47 revealed an aneuploid karyotype with complex abnormalities including 1q+, 2p+, and 14q+. To our knowledge, this is the only IgD-secreting myeloma cell line and would provide a useful tool for the study of IgD production and IgD myeloma.


Transient reductions in serum cholesterol after renal transplantation. Declines in serum cholesterol have been reported in patients with altered immune system activity. However, the frequency and clinical significance of transient reductions in serum cholesterol after renal transplantation are unknown. In the present retrospective study, we examined the frequency and clinical setting of reduced serum cholesterol (< or = 4.40 mmol/L [170 mg/dL]) in patients who each had 28 +/- 7 (total, 1,110) cholesterol determinations during the first year posttransplant. Reduced cholesterol was found on at least one occasion in 26 of 40 (65%) patients. Ninety-two percent (119/129) of the reduced cholesterol values occurred in one of three clinical settings: (1) within 10 days after transplantation, (2) within 6 weeks before or after the onset of acute rejection, or (3) within 6 weeks before or after the onset of a cytomegalovirus infection (CMV). Multiple linear regression analysis showed that the relationship between reductions in cholesterol associated with acute rejection was independent of CMV and the type of immunosuppression (one half of the patients were treated with cyclosporine [CSA]). The fact that serum albumin was reduced during CMV, but not during acute rejection, suggested that reduced cholesterol associated with rejection was relatively specific, and was not caused by a generalized leak of plasma proteins or by poor nutrition. Thus, during the first year posttransplant, reductions in serum cholesterol are most often associated with acute rejection episodes and/or CMV.(ABSTRACT TRUNCATED AT 250 WORDS)


The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa. The finding of a mutation in the beta subunit of the cyclic GMP (cGMP) phosphodiesterase gene causing retinal degeneration in mice (the Pdeb gene) prompted a search for disease-causing mutations in the human phosphodiesterase gene (PDEB gene) in patients with retinitis pigmentosa. All 22 exons including 196 bp of the 5' region of the PDEB gene have been assessed for mutations by using single-strand conformational polymorphism analysis in 14 patients from 13 unrelated families with autosomal recessive retinitis pigmentosa (ARRP). No disease-causing mutations were found in this group of affected individuals of seven different ancestries. However, a frequent intronic and two exonic polymorphisms (Leu489----Gln and Gly842----Gly) were identified. Segregation analysis using these polymorphic sites excludes linkage of ARRP to the PDEB gene in a family with two affected children.


SSCP and segregation analysis of the human type X collagen gene (COL10A1) in heritable forms of chondrodysplasia. Type X collagen is a homotrimeric, short chain, nonfibrillar collagen that is expressed exclusively by hypertrophic chondrocytes at the sites of endochondral ossification. The distribution and pattern of expression of the type X collagen gene (COL10A1) suggests that mutations altering the structure and synthesis of the protein may be responsible for causing heritable forms of chondrodysplasia. We investigated whether mutations within the human COL10A1 gene were responsible for causing the disorders achondroplasia, hypochondroplasia, pseudoachondroplasia, and thanatophoric dysplasia, by analyzing the coding regions of the gene by using PCR and the single-stranded conformational polymorphism technique. By this approach, seven sequence changes were identified within and flanking the coding regions of the gene of the affected persons. We demonstrated that six of these sequence changes were not responsible for causing these forms of chondrodysplasia but were polymorphic in nature. The sequence changes were used to demonstrate discordant segregation between the COL10A1 locus and achondroplasia and pseudoachondroplasia, in nuclear families. This lack of segregation suggests that mutations within or near the COL10A1 locus are not responsible for these disorders. The seventh sequence change resulted in a valine-to-methionine substitution in the carboxyl-terminal domain of the molecule and was identified in only two hypochondroplasic individuals from a single family. Segregation analysis in this family was inconclusive, and the significance of this substitution remains uncertain.


Electron microscopy analysis of mineral fibers in human lung tissue. In the present study, lung samples from 126 autopsied cases were examined to determine the content of mineral fibers using analytical transmission electron microscopy (ATEM). The cases were divided into four groups (22 lungs of persons exposed to ambient environmental pollution, 32 cases of mesothelioma, 38 cases of primary lung cancer, and 34 asbestosis cases, 13 of these with additional pleural plaques). Fibers were counted, measured, and mineralogically identified using a combination of X-ray microanalysis and electron diffraction of the non-oriented fiber. Concentration of fibrous particles (defined as particles above 1 micron in length with roughly parallel long sides and an aspect ratio of 5:1 and greater) was calculated as fibers 10(6)/g dry lung weight. The concentration of chrysotile was found to be similar throughout the groups except for two cases in the asbestosis group with comparably high numbers of chrysotile. However, a remarkable difference for amphiboles could be observed between the groups. Asbestos bodies were mostly found in the asbestosis group. There was a rather good correlation between numbers of amphibole fibers and asbestos bodies, with an average ratio of 10:1. For comparison purposes between occupationally exposed/non-exposed individuals, a transition was found in the concentration range of 3-10(7) asbestos fibers/g dried lung weight.


Small airways function of silica-exposed workers. Small airways obstruction may be present for many years before chronic airway obstruction becomes evident. Several spirometric indices, especially flow rates at low lung volumes, may reflect the status of small airways. Time domain indices, by using moments analysis of the volume time spirogram, have also been shown to be sensitive indicators of small airways obstruction. In this study, we have applied the various spirometric indices as well as time domain indices to a group of granite quarry workers without radiographic evidence of silicosis or physiological evidence of obstruction to the larger airways. The aim was to evaluate small airways function in relation to dust exposure in subjects with normal ratio of the forced expiratory volume in one second to the forced vital capacity (FEV1/FVC) and normal FVC. The volume-time spirograms of 140 quarry workers were digitized using an electronic digitizer connected to a microcomputer where flow and time domain indices were computed. The workers were divided into three exposure groups based on their occupational history. With adjustment for age, height, and smoking status, all the time domain indices showed significant small airways obstruction with increasing dust exposure. Smokers had greater degree of airways obstruction than the non-smokers, with a similar trend of increase in small airways obstruction in relation to higher exposure. Our present study suggests that small airways obstruction is present among silica exposed workers in the absence of radiological evidence of silicosis and large airways obstruction. There was also evidence of increasing small airways obstruction in higher dust exposure group. Our study also suggests that time domain indices are more sensitive to small airways obstruction.


Painful diffuse osteosclerosis after intravenous drug abuse. We identify a new syndrome of acquired painful diffuse osteosclerosis associated with past intravenous drug abuse in two adults. A 28-year-old white woman and a 38-year-old black man with a history of non-A, non-B chronic active hepatitis were referred to us for increasing bone pain that was especially severe in their lower extremities. They were studied at our clinical research center. Skeletal radiographs documented progressive generalized osteosclerosis. Increased bone mass was confirmed by dual-energy radiography, and bone scintigraphy showed diffusely increased radionuclide accumulation. Serum biochemical studies revealed elevated alkaline phosphatase activity and osteocalcin levels, mild to moderately increased 1,25-dihydroxyvitamin D concentrations, and normal parathyroid hormone levels. In urine, hydroxyproline excretion was elevated, whereas calcium levels were reduced. Iliac crest histomorphometry showed increased rates of bone formation. Hematology, renal function, serum protein electrophoresis, and screening for fluorosis as well as vitamin A and heavy metal poisoning were all normal. Family histories were negative. Both patients were seropositive for antibody against hepatitis C virus as well as against Epstein-Barr virus (antiviral capsid antigen IgG but not IgM). Each subject was seronegative for cytomegalovirus, human immunodeficiency virus (HIV) 1 and 2, and human T-cell lymphotropic virus (HTLV) 1 and 2. Assay for reverse transcriptase in lymphocyte co-culture fluid and polymerase chain reaction studies using HIV-1 primers on peripheral monocyte DNA were negative. Treatment with synthetic salmon calcitonin in both individuals rapidly led to decreased bone pain and to a decline in biochemical parameters of accelerated bone turnover. Painful diffuse osteosclerosis can follow intravenous drug abuse and is possibly caused by parenteral transmission of a virus that in some way stimulates bone formation.


Risk of hepatoblastoma in familial adenomatous polyposis. Infantile and childhood hepatoblastoma occurs more frequently in persons heterozygous for the familial adenomatous polyposis (FAP) gene than in the general population. This observation is based on numerous case reports plus the results of an international survey of FAP registries. However, the frequency of this rare tumor in FAP patients is unknown. In a retrospective review of our family history data, 2/470 (0.42%) children born to 241 patients with FAP had hepatoblastoma. This figure is significantly higher than the 1/100,000 incidence of hepatoblastoma in the general population. However, for genetic counseling purposes, an empiric risk of less than 1% for hepatoblastoma can be cited to persons with FAP for their children.


Abnormalities of erythrocyte sodium transport systems in Bartter's syndrome. The basic tubular alteration present in Bartter's syndrome is still a subject of controversy. The possibility that a generalized defect in the transmembrane ion transport underlies the disease has been extensively investigated. Previous evaluations of cellular sodium metabolism in Bartter patients showed extremely variable findings. We have examined in the red blood cells of two patients with Bartter's syndrome the intracellular Na+ and K+ concentrations, the activity of the ouabain-sensitive Na+/K+ pump, furosemide-sensitive Na+/K+ cotransport, Na+/Li+ countertransport and the rate constant of Na+ and K+ passive permeability. We have compared these values with those of healthy subjects and patients with chronic hypokalemia produced by conditions other than Bartter's syndrome. Ouabain-sensitive Na+/K+ pump activity was decreased in both patients, whereas Na+/Li+ countertransport was activated. One of the patients also exhibited markedly decreased intraerythrocyte K+ concentration and decreased furosemide-sensitive Na+/K+ cotransport. The other had increased Na+/K+ cotransport activity and Na+ passive permeability. Intracellular Na+ and passive permeability to K+ were normal in both subjects. Neither oral potassium supplementation (100 mEq/day) nor indomethacin treatment (150 mg/day) could correct these abnormalities. Our results are partially consistent with previous observations and indicate the existence of heterogenous abnormalities of erythrocyte sodium transport systems in patients with Bartter's syndrome which are not a consequence of chronic hypokalemia.


Verapamil reverses glucose intolerance in preexisting chronic renal failure: studies on mechanisms. Glucose-induced insulin secretion is impaired in chronic renal failure (CRF), and this abnormality is due to the elevation of cytosolic calcium [Ca2+]i and other derangements in pancreatic islet metabolism. Verapamil given to rats from day 1 of CRF prevented the rise in [Ca2+]i of islets and the impairment in insulin secretion. However, it is not known whether verapamil can reverse the abnormalities of islet function and metabolism in animals with preexisting renal failure. Such a documentation has important clinical implications for the treatment of carbohydrate intolerance in patients with CRF. The present study examined this question. After 6 weeks of CRF, rats were randomized into two subgroups and maintained for additional 6 weeks. One subgroup received intraperitoneal injections of verapamil (0.1 micrograms/kg body weight twice daily) and the other received vehicle only. At the time of randomization, there were no significant differences between the two subgroups in their body weight, plasma levels of calcium, phosphorus and creatinine, serum parathyroid hormone and creatinine clearance. Similarly, at the time of sacrifice (12 weeks), there were no significant differences in these parameters except for a modestly lower plasma level of creatinine and modestly higher creatinine clearance.(ABSTRACT TRUNCATED AT 250 WORDS)


Properties of an endogenous arachidonic acid--elicited relaxing mechanism in human placental vessels. Our objective was to examine the action of arachidonic acid on tone in isolated human placental arteries and veins (1 to 2 mm diameter) for mechanisms involving endothelium-derived mediators, the stimulation of guanylate cyclase, and prostaglandin and cytochrome P450 metabolites. Pharmacologic probes and endothelium-removal were used to examine the mechanism of relaxation to arachidonic acid (10 nmol/L to 10 mumol/L) observed in placental arteries and veins obtained after delivery from uncomplicated term pregnancies and precontracted with prostaglandin F2 alpha. Neither removal of the endothelium nor inhibition of prostaglandin biosynthesis with 10 mumol/L indomethacin, arginine-derived nitric oxide formation with 30 mumol/L nitro-L-arginine, or guanylate cyclase stimulation with 10 mumol/L LY83583 altered the observed relaxation to arachidonic acid in either preparation. However, this relaxation was markedly attenuated by 30 mumol/L proadifen (SKF-525A), an inhibitor of cytochrome P450, suggestive of a role for arachidonic acid metabolism by an epoxygenase or monooxygenase pathway. Human placental arteries and veins possess an endogenous mechanism of relaxation to arachidonic acid, which seems to be mediated by metabolites formed by a cytochrome P450 enzyme. This endogenous relaxation mechanism could function as a suppressor of vasospasm in the placental circulation.


Magnesium sulfate therapy in preeclampsia is associated with increased urinary cyclic guanosine monophosphate excretion. Our objective was to determine if maternal urinary cyclic guanosine monophosphate levels are altered in preeclampsia. Aliquots from 24-hour urine samples collected from 57 women with preeclampsia and 14 normotensive pregnant women in the third trimester of pregnancy were assayed for urinary cyclic guanosine monophosphate. Urinary cyclic guanosine monophosphate values were expressed per milligram of urinary creatinine to standardize for renal function. There was no difference in gestational age at time of urine collection between the two groups. Urinary cyclic guanosine monophosphate levels (mean +/- SD) were similar between normotensive and preeclamptic pregnant women (751 +/- 498 vs 632 +/- 363 pmol/mg urinary creatinine, respectively, p = 0.12). Preeclamptic women receiving magnesium sulfate had significantly higher levels of urinary cyclic guanosine monophosphate than those not receiving magnesium sulfate (786 +/- 360 vs 555 +/- 344 pmol/mg urinary creatinine, respectively, p = 0.02). These preliminary results indicated that cyclic guanosine monophosphate excretion increases in patients with preeclampsia during magnesium sulfate infusion. The vascular smooth muscle relaxation effects of magnesium sulfate may be mediated by directly increasing cyclic guanosine monophosphate production or indirectly through endothelium-derived relaxing factor.


Phenotype of villous stromal cells in placentas with cytomegalovirus, syphilis, and nonspecific villitis. Villous stromal cells (VSC) play an important role in fetomaternal placental immune function. We studied the phenotype of VSC in infection by cytomegalovirus (CMV) and syphilis as well as nonspecific villitis and compared the findings with gestational age-matched controls. Monoclonal antibodies directed against total leukocytes, T cells, B cells, macrophages, dendritic cells, granulocytes and HLA-DR as well as polyclonal antibodies against S-100, alpha-1 antichymotrypsin, and lysozyme were used. In controls, the immunocytochemical response for each marker was either negative or weakly positive. In contrast, the VSC in CMV-infected and nonspecific villitis showed intense reactivity to various macrophage markers. In syphilis, reactivity with macrophage markers such as lysozyme and MAC387 were weaker, and reactivity to HLA-DR and S-100 was much stronger. Endothelial cells strongly expressed the monocyte/granulocyte marker CD15 in the diseased states, especially in syphilis, relative to controls. We conclude that the phenotype of VSC is altered in disease states and that the changes are dependent to some degree on the specific subset of chronic villitis.


Localization of cellular retinoid-binding proteins in human cervical intraepithelial neoplasia and invasive carcinoma. Cellular retinoic acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP) were localized in biopsies of normal squamous epithelium, cervical intraepithelial neoplasia (CIN), and invasive squamous cell cancer of the cervix uteri by immunohistochemistry. In both the normal stratified squamous epithelium of the exocervix and low-grade CIN, CRABP I was present predominantly in the basal layer of the epithelium. The more superficial, differentiated cell layers lacked immunoreactive protein. In high-grade CIN (CIN2-3), the distribution of CRABP I was altered. Immunoreactive CRABP I was detected in all layers of high-grade CIN. In squamous cell carcinoma of the cervix, CRABP I was detected in cells throughout the tumor but was minimal in cells demonstrating squamous differentiation. In contrast to CRABP I, CRBP was diffusely present throughout the cervical epithelium irrespective of the state of differentiation or the presence of disease.


Regulation by calcitonin of Na(+)-K(+)-Cl- cotransport in a rabbit thick ascending limb cell line. The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.


Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP. Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)


Beta 2-adrenoceptor density in fibroblast culture correlates with human NaCl sensitivity. To study salt sensitivity in humans and its relation to expression of adrenoceptors, 20 male normotensive Caucasians were investigated on a diet of 180 mmol NaCl/day followed by 60 mmol NaCl/day over 2 wk and again by 180 mmol NaCl/day over 2 wk, and blood pressure changes were assessed by long-term oscillatory blood pressure monitoring under basal conditions. Individual cell cultures of skin fibroblasts from skin biopsies were also established, and alpha 2- and beta 2-adrenoceptors were measured. Seven subjects were salt sensitive, and the remainder were salt resistant. Cultured skin fibroblasts in salt-sensitive subjects express less than half the number of beta 2-adrenoceptors compared with salt-resistant subjects (65 +/- 12.7 vs. 173 +/- 14.8 fmol/mg, P less than 0.001), and there is a correlation between the absolute rise of blood pressure on a high-salt diet and the density of beta 2-adrenoceptors (r = -0.67, P less than 0.01). It remains to be established whether a reduced in vitro density of beta 2-adrenoceptors in cultured cells is causally related to salt sensitivity in normotensive humans.


Protein kinase A phosphorylation enhances sodium channel currents in Xenopus oocytes. The voltage-sensitive rat brain sodium channel is known to be phosphorylated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA), but the functional significance of that phosphorylation is unknown. We have shown that rat brain sodium channel currents expressed in Xenopus oocytes were enhanced by induction of PKA activity. Stimulation of the beta 2-adrenergic receptor or treatment with dibutyryl cAMP resulted in increased sodium current amplitudes without affecting the voltage dependence of channel activation or inactivation. These increases were completely blocked by preinjection of protein kinase inhibitor, a specific inhibitor of PKA. Injection of phosphatase into the oocytes resulted in a significant decrease in sodium current amplitude, indicating that phosphorylation is important for basal levels of sodium channel activity in oocytes. The enhancement was specific for the rat brain IIA sodium channel, because currents expressed from the rat muscle microI sodium channel were not enhanced by the same procedures. These data demonstrate a modulatory role of PKA phosphorylation on brain sodium channel function and suggest a means by which the electrical excitability of cells may be regulated.


Abnormalities in cardiac alpha 1-adrenoceptor and its signal transduction in streptozocin-induced diabetic rats. To investigate a mechanism of diabetic cardiomyopathy, we examined an alteration of cardiac alpha 1-adrenoceptor (alpha 1-AR) signaling in streptozotocin-induced diabetic rats. In diabetes, the cell surface alpha 1-AR concentration on isolated cardiac myocytes decreased by 45% without any change in the dissociation constant, and, moreover, norepinephrine (NE)-stimulated ventricular inositol 1,4,5-trisphosphate (IP3) production was also decreased by 34%. In contrast, basal ventricular protein kinase C (PKC) activity was elevated in both cytosolic (by 98%) and membrane (by 41%) fractions in diabetes. All of these abnormalities seen in diabetes were reversed by chronic insulin treatment. Rapid activation of PKC by phorbol ester in the normal rat heart revealed decreases in both receptor number (by 19%) and NE-stimulated IP3 production (by 21%). These results indicate that the impairment of cardiac alpha 1-AR signaling is closely associated with the diabetic state and may be linked, at least in part, with the abnormal activation of cardiac PKC.


Interleukin-1-induced corticosterone release occurs by an adrenergic mechanism from rat adrenal gland. Interleukin-1 (IL-1) has been shown to stimulate corticosterone release from the adrenal gland directly, and indirectly through activation of the hypothalamic-pituitary-adrenal axis. The aim of this paper was to determine whether IL-1-stimulated corticosterone release occurs indirectly through the local release of catecholamines from the rat adrenal gland. To accomplish this, experiments were conducted on both quartered rat adrenal glands and primary cultures of dispersed adrenal cells. Incubation of quartered adrenals with adrenocorticotropic hormone (ACTH, 10(-12) to 10(-8) M) or IL-1 beta (10(-12) to 10(-8) M) resulted in dose-dependent increases (P less than 0.05) in corticosterone release. Corticosterone release stimulated by 10(-8) M doses of ACTH and IL-1 beta began to rise 30 min after incubation and peaked at 2 h. In primary cultures of adrenal cells, IL-1 alpha and IL-1 beta elevated corticosterone release after a 24-h incubation period. ACTH elevated corticosterone levels at 4 and 24 h. The stimulatory effect of IL-1 on corticosterone release was mimicked by epinephrine (10(-6) M), and was selectively blocked by the alpha-adrenergic antagonist phentolamine (10(-5) M). The beta-adrenergic antagonist propranolol (10(-5) M) did not change IL-1-induced corticosterone release. Neither phentolamine nor propranolol had an effect on ACTH-stimulated corticosterone release. Both IL-1 alpha and IL-1 beta significantly increased (P less than 0.05) epinephrine levels after a 24-h incubation period compared with media-treated controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibitors of Na(+)-H+ exchange block stimulus-provoked pineal melatonin synthesis. In rat pinealocytes, amiloride can modulate adrenergic-stimulated cyclic nucleotide accumulation. In this study, the effect of amiloride on melatonin production was characterized. Addition of 5-(N,N-hexamethylene)amiloride, a potent inhibitor of the Na(+)-H+ antiport, dose dependently inhibited norepinephrine- and isoproterenol-stimulated N-acetyltransferase (NAT) activity and melatonin production. Similar inhibition was also observed when pineal melatonin synthesis was stimulated directly with forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that the site of inhibition is distal to cAMP accumulation. Similarities between the inhibitory potencies of amiloride derivatives on the Na(+)-H+ antiport and pineal melatonin synthesis indicate that the observed inhibition on pineal melatonin synthesis by amilorides may be secondary to their actions on the Na(+)-H+ antiport. Further studies also indicate that the inhibitory effect of amilorides was not secondary to its cytotoxic actions and that amilorides had no direct antagonistic action on NAT activity. Our findings, therefore, suggest that, in addition to their effects on cyclic nucleotide accumulation, the Na(+)-H+ antiport also plays an important role in the cAMP-mediated melatonin synthesis in the rat pineal gland.


Effects of ovarian hormones on brain opioid binding sites in castrated female rats. These experiments were performed to analyze whether treatments of ovariectomized female rats with ovarian steroid regimens able to induce either an increase (positive feedback effect) or a decrease (negative feedback effect) of serum levels of luteinizing hormone (LH) have some impact on the characteristics of mu-opioid binding sites in circumscribed areas of the brain. The increase of serum levels of LH elicited by a treatment with estradiol benzoate (EB) plus progesterone (P; positive feedback effect) was accompanied by a significant decrease in the number of mu-binding sites in the hypothalamus and in the corpus striatum. The decrease in serum levels of LH induced by a treatment with EB alone (negative feedback effect) brought about a significant increase of the number of mu-binding sites in the thalamus and in the hippocampus. These results seem to suggest that the release of LH induced by EB plus P may involve a decrease of hypothalamic mu-binding sites. Apparently, the inhibitory effect on LH release exerted by EB alone does not involve any change of the density of these binding sites in the hypothalamus.


A primary role for protein kinase A in smooth muscle relaxation induced by adrenergic agonists and neuropeptides. Many studies suggest that smooth muscle relaxation caused by beta-adrenergic agents and various neuropeptides occurs as a result of an increase in cellular adenosine 3',5'-cyclic monophosphate (cAMP). However, the evidence is indirect, and furthermore does not demonstrate that an increase in cAMP is essential for mediating relaxation. To define more clearly the role of cAMP in receptor-mediated smooth muscle relaxation, we used a specific competitive antagonist of the action of cAMP on protein kinase A, (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], and its S isomer, (S)-p-cAMPS, which functions as a cAMP agonist. In gastric smooth muscle cells from guinea pig, (S)-p-cAMPS caused a dose-related relaxation [50% inhibitory concentration (IC50) 86 +/- 59 nM]. Vasoactive intestinal peptide (VIP) produced smooth muscle cell relaxation (IC50 2.3 +/- 0.8 nM) through occupation of specific VIP receptors. (R)-p-cAMPS inhibited VIP-induced relaxation, with a rightward shift in the VIP dose-response curve, suggesting competitive antagonism. Furthermore, (R)-p-cAMPS inhibited relaxation induced by other agents that increase cellular cAMP (isoproterenol, calcitonin gene-related peptide, and glucagon) but not that induced by ATP or sodium nitroprusside. (R)-p-cAMPS had no effect on contraction stimulated by carbachol, cholecystokinin, or substance P. These data demonstrate that activation of protein kinase A is primarily responsible for mediating gastrin smooth muscle relaxation produced by adrenergic agents and various neuropeptides.


Characterization of colonic circular smooth muscle cells in culture. The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Rabbit esophageal cell cytoplasmic pH regulation: role of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mtext>Na</mml:mtext><mml:mo>+</mml:mo></mml:msup></mml:math>-<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mtext>H</mml:mtext><mml:mo>+</mml:mo></mml:msup></mml:math> antiport and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mtext>H</mml:mtext><mml:mo>+</mml:mo></mml:msup></mml:math>-dependent <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mtext>H</mml:mtext><mml:mtext>C</mml:mtext><mml:msubsup><mml:mtext>O</mml:mtext><mml:mn>3</mml:mn><mml:mo>-</mml:mo></mml:msubsup></mml:math> tra. Regulation of cytoplasmic pH (pHi) of esophageal cells assumes importance as these cells can be exposed to mucosally absorbed acid during gastroesophageal reflux episodes. In this study, we examined whether esophageal cells possess pHi transport systems. Esophageal cells were harvested utilizing a gentle trypsin technique that yielded <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mn2</mml:mn<mml:mo-</mml:mo<mml:mn5</mml:mn<mml:mo×</mml:mo<mml:msup<mml:mn10</mml:mn<mml:mn6</mml:mn</mml:msup</mml:math cells per esophagus. Cells were attached to a glass cover slip that had been pretreated with rat-tail collagen, and pHi was measured continuously in a spectrofluorometer utilizing 2',7'-bis(2-carboxyethyl)-5(-6)- carboxyfluoroscein acetoxymethyl ester as a pH-sensitive fluorescent probe. <DispFormula<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mtextH</mml:mtext<mml:mo+</mml:mo</mml:msup</mml:math</DispFormula The basal pHi of cells exposed to a <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mtextH</mml:mtext<mml:mo+</mml:mo</mml:msup</mml:math-containing solution averaged 7.52 ± 0.20 (n = 6). The pHi declined slightly but not significantly to 7.46 ± 0.12 with the addition of 5% <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mtextC</mml:mtext<mml:msub<mml:mtextO</mml:mtext<mml:mn2</mml:mn</mml:msub</mml:math and 28 mM <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mtextNa</mml:mtext<mml:mtextH</mml:mtext<mml:mtextC</mml:mtext<mml:msub<mml:mtextO</mml:mtext<mml:mn3</mml:mn</mml:msub</mml:math When H2 4,4'-diisothiocyanatostilbene- 2,2'-disulfonic acid (DIDS; 0.5 mM) was added, pHi was unchanged. However, addition of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mn10</mml:mn<mml:mn-6</mml:mn</mml:msup</mml:math M amiloride caused pHi to decrease to 7.29 ± 0.18 (P less than 0.01). When cells were acidified (pHi 6.3-7.0) using a<DispFormula<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mrow<mml:msup<mml:mia</mml:mi<mml:mn2</mml:mn</mml:msup<mml:mo+</mml:mo<mml:msup<mml:mib</mml:mi<mml:mn2</mml:mn</mml:msup<mml:mo=</mml:mo<mml:msup<mml:mic</mml:mi<mml:mn2</mml:mn</mml:msup</mml:mrow</mml:math</DispFormula(20 mM) pulse technique, pHi was rapidly restored toward neutrality in the presence of a <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mtextH</mml:mtext<mml:mtextC</mml:mtext<mml:msubsup<mml:mtextO</mml:mtext<mml:mn3</mml:mn<mml:mo-</mml:mo</mml:msubsup</mml:math-free external <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mtextNa</mml:mtext<mml:mo+</mml:mo</mml:msup</mml:math concentration ([<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mtextNa</mml:mtext<mml:mo+</mml:mo</mml:msup</mml:math]o)-containing solution (pH units/min = 0.26 ± 0.12; n = 8). Alkalinization was completely blocked with <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mn10</mml:mn<mml:mn-6</mml:mn</mml:msup</mml:math M amiloride. In the presence of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:msup<mml:mn10</mml:mn<mml:mn-6</mml:mn</mml:msup</mml:math M amiloride, 28 mM <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mtextNa</mml:mtext<mml:mtextH</mml:mtext<mml:mtextC</mml:mtext<mml:msub<mml:mtextO</mml:mtext<mml:mn3</mml:mn</mml:msub</mml:math, and 5% <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"<mml:mtextC</mml:mtext<mml:msub<mml:mtextO</mml:mtext<mml:mn2</mml:mn</mml:msub</mml:math, acidified cells also alkalinized, although at a slower rate (0.11 ± 0.04 pH units/min; n = 16).(ABSTRACT TRUNCATE


Atriopeptin-induced increases in endothelial cell permeability are associated with elevated cGMP levels. To investigate the mechanism of nonrenal capillary hyperfiltration, we studied the effect of atriopeptin (AP) III and AP I on permeability and intracellular cyclic nucleotide levels in cultured bovine pulmonary artery endothelial cell monolayers. Permeability to albumin was assessed by the albumin transfer rate across endothelial cell monolayers, following a 4-h incubation with atriopeptins. AP III (0.01, 0.1, and 1 microM) caused a concentration-dependent increase in the albumin transfer rate. AP III induced a threefold increase in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels during the incubation period. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), enhanced the AP III-induced increase in permeability and cGMP accumulation by 16-fold at maximum. 8-Bromoguanosine 3',5'-cyclic monophosphate, a hydrolysis-resistant cGMP analogue, caused a slight but significant increase in permeability. In contrast, AP I, a weak agonist of the cGMP-coupled ANP receptor, did not elicit an increase in permeability at concentrations of 0.1 and 1 microM. Although AP I (1 microM) caused a significant increase in cGMP by 33 and 60% in the absence and presence of IBMX, the increase was markedly less compared with AP III. AP III did not cause a change in intracellular cAMP levels during the incubation period. These observations suggest that in our system AP III increases the permeability of endothelial cell monolayers in association with an elevated cGMP level. Thus an increase in permeability might be involved in the mechanism of ANP-induced capillary hyperfiltration.


Circulating xanthine oxidase mediates lung neutrophil sequestration after intestinal ischemia-reperfusion. Injury to nonpulmonary organ systems often initiates systemic processes that cause recruitment of neutrophils to the lung. We found that rats subjected to intestinal ischemia-reperfusion (I/R) had increased transvascular leak of 125I-labeled albumin into lungs and decreased lung ATP levels (P less than 0.05). In addition, rats subjected to intestinal I/R had increased plasma xanthine oxidase (XO) activity, plasma leukotactic activity for neutrophils, and lung neutrophil retention (assessed by morphometry and myeloperoxidase activity) compared with sham-treated rats (P less than 0.05). By comparison, after intestinal I/R, rats fed an allopurinol- or tungsten-enriched diet had decreased plasma and intestinal XO activities, decreased plasma leukotacic and lung myeloperoxidase (MPO) activities, decreased lung leak, and increased lung ATP levels compared with rats fed control diets (P less than 0.05). Further studies suggested a more specific role for circulating rather than tissue XO in mediating lung neutrophil accumulation but not lung leak. Plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, increased in rats administered purified XO intravenously. In addition, plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, decreased in rats administered antisera against XO and then subjected to intestinal I/R. We conclude that circulating XO increases acutely and may contribute to pulmonary retention of neutrophils after an ischemic intestinal insult.


Effects of hydrogen peroxide on the responsiveness of isolated canine bronchi: role of prostaglandin E2 and I2. The present study was design to determine the role of prostaglandin E2 and I2 in the responses of isolated canine airways to H2O2. Rings of canine third-order bronchi, some of which had undergone mechanical denudation of the epithelium, were suspended in organ chambers; isometric tension was recorded. During contractions to acetylcholine, H2O2 induced concentration-dependent relaxations. The relaxations were attenuated significantly by indomethacin, acetylsalicylic acid, and methylene blue. H2O2 increased the release of prostaglandin E2 and 6-keto-prostaglandin F1 alpha and the content of adenosine 3',5'-cyclic monophosphate (cAMP). These effects were abolished by indomethacin or methylene blue. H2O2 did not affect the content of guanosine 3',5'-cyclic monophosphate significantly. These observations suggest that 1) H2O2 relaxes canine bronchial smooth muscle and 2) elevation of tissue content of cAMP induced by prostaglandin E2 and I2 may be involved. These phenomena did not appear to be modulated by the respiratory epithelium, since H2O2-induced relaxations and increases in the release of PGE2 and 6-ketoprostaglandin F1 alpha were similar in preparations with and without epithelium. However, after treatment with methylene blue, H2O2 induced contractions only in preparations with epithelium. These epithelium-dependent contractions were not affected by inhibitors of cyclooxygenase and lipoxygenase.


Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6). Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.


Glucocorticoids inhibit colonic aldosterone-induced conductive Na+ absorption in adrenalectomized rat. Low-dose glucocorticoids induce only electroneutral Na absorption in rat colon. High-dose dexamethasone induces both electroneutral and electrogenic Na absorption. Aldosterone induces only electrogenic Na absorption and inhibits basal electroneutral transport in distal colon. To define the interrelationship between glucocorticoid-mediated and aldosterone-mediated Na absorption, adrenalectomized rats were treated with aldosterone and either glucocorticoid-receptor-specific doses of dexamethasone or the specific glucocorticoid RU26988. In combination the steroids did not increase Na and Cl absorption in proximal and distal colon and transmural potential difference (PD) and K secretion in distal colon as much as aldosterone alone. Na absorption was not inhibited by spironolactone or amiloride (10(-4) M). Transport in both segments was by Na-H exchange as demonstrated by marked inhibition by amiloride (10(-3) M) and the Na-H antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride. Thus glucocorticoids not only decreased Na absorption but also produced a marked qualitative change in the mode of Na absorption in aldosterone-treated rats. Acute dexamethasone infusion in rats pretreated with aldosterone decreased Na absorption and transmural PD within 30 min, suggesting inhibition of electrogenic Na absorption at a step distal to synthesis of aldosterone-induced proteins. These findings suggest that upregulation of one Na absorptive mechanism downregulates the other. This may explain why in intact unstressed rats there is little or no conductive Na absorption, despite sufficient endogenous steroid to occupy the aldosterone receptor. It may also explain why in proximal colon of intact rats, despite the presence of aldosterone receptors, even prolonged aldosterone exposure does not induce significant conductive Na absorption.


Cytoskeleton disruption and apical redistribution of proximal tubule Na(+)-K(+)-ATPase during ischemia. The polar distribution of Na(+)-K(+)-ATPase to the basolateral membrane of proximal tubule cells is essential for the efficient and vectorial reabsorption of Na+ and may be dependent on the formation of a metabolically stable, detergent-insoluble complex of Na(+)-K(+)-ATPase with the actin membrane cytoskeleton. The present studies utilized immunocytochemical techniques to demonstrate and quantify the apical redistribution of Na(+)-K(+)-ATPase during mild ischemia (15 min) that occurred in proximal (1.3 +/- 0.9 vs. 4.5 +/- 1.1 particles/100 microns surface membrane, P less than 0.01) but not distal tubule cells. Treatment of control apical membranes with 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C), a fluidizing agent, markedly increased membrane fluidity without any effect on Na(+)-K(+)-ATPase activity. In brush-border membrane vesicles isolated after ischemia, however, A2C further increased an already elevated Na(+)-K(+)-ATPase activity. During ischemia, total cellular Na(+)-K(+)-ATPase activity remained unaltered, but the Triton X-100-soluble (noncytoskeleton associated) fraction of Na(+)-K(+)-ATPase increased significantly following 15 and 30 min. There was a corresponding decrease in the Triton X-100-insoluble fraction of Na(+)-K(+)-ATPase, with the ratio of detergent-soluble to -insoluble Na(+)-K(+)-ATPase increasing from 13 +/- 2 to 32 +/- 5% (P less than 0.01) during 30 min of ischemia. Western blot analysis of the Triton X-100-soluble fraction, following 30 min of ischemic injury, revealed the presence of Na(+)-K(+)-ATPase, actin, fodrin, and uvomorulin. However, in a fraction highly enriched for Na(+)-K(+)-ATPase, neither actin, fodrin, nor uvomorulin was detected.(ABSTRACT TRUNCATED AT 250 WORDS)


Difference in effect of atrial natriuretic peptide on cGMP in aortic and coronary smooth muscle cells. Blood vessels show a heterogeneous response to the atrial natriuretic peptide (ANP). In our experiments thoracic aorta from the guinea pig relaxed in response to atriopeptin III (AP; rat ANP-103-126) and to sodium nitroprusside (SNP). In contrast, in perfused guinea pig hearts, AP III produced no change in coronary flow, while SNP increased flow. In smooth muscle cells cultured from the coronary system (CASM) and from the thoracic aorta (TASM), we compared receptor binding and the effects on guanosine 3',5'-cyclic monophosphate (cGMP) production of AP III. AP III bound specifically with equal affinity and with equivalent numbers of binding sites in both cell types. AP III produced a dose-dependent increase in cGMP in TASM (50% effective concentration approximately 3 nM) with a maximum 11-fold increase over basal at 1 microM AP III. In contrast, in CASM, AP III failed to increase cGMP. Nitroprusside increased cGMP in both cell types. Autoradiograms of 125I-labeled AP III linked to cell membranes showed bands at 70 kDa (ANP-C receptor) in both cell types. A second band at 140 kDa (ANP-B receptor) was only seen in TASM. These results suggest that smooth muscle cells of coronary resistance vessels of the guinea pig do not express the particulate guanylyl cyclase that is activated by ANP.


Effects of Na-K-ATPase inhibition on catecholamine reactivity in rat pulmonary artery. The objective of this study was to determine the effects of Na-K-adenosinetriphosphatase (ATPase) inhibition on aspects of vascular reactivity in rat pulmonary artery, including resting tone and catecholamine reactivity, loss of reactivity to repeated catecholamine stimulation, and contraction-relaxation kinetics. The effect of ATPase inhibition on resting tone and catecholamine reactivity was determined by comparing the responses to 10(-4) and 10(-3) M ouabain (Oua-4, Oua-3), 10(-4) M tyramine, and 10(-6) M norepinephrine (NE) pre- and postouabain. Loss of responsiveness to NE was induced by repeated cumulative doses of NE (NE I, NE II) with 60 min between exposures. ATPase inhibition was superimposed on this loss of reactivity by 1) administration of Oua-4 or Oua-3 15 min before NE II, 2) incubation in K(+)-free solution 15 min before NE II, and 3) incubation in K(+)-free solution 15 min before NE I for the entire protocol. The effect of ATPase inhibition on the response pattern after repetitive dosing with phenylephrine (PE) and KCl was also determined. To correlate ATPase activity with the loss of reactivity after catecholamine stimulation, we measured ATPase activity (86Rb uptake) at different time points after repeated NE administration. The effect of ATPase inhibition on contraction-relaxation rates after catecholamine stimulation was determined by analyzing the response to repeated single doses of NE in normal salt solution and after incubation in K(+)-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic coronary arterial stenosis impairs alpha 1-adrenoreceptor signaling and cardiac performance in rats. Coronary stenosis was induced in rats to determine whether chronic coronary artery constriction resulted in impairment of cardiac pump performance, alterations in alpha 1-adrenoreceptor signal transduction, and inadequate myocardial hypertrophy, and these parameters were examined 6 mo later. A 50% reduction in coronary diameter was associated with an elevation in left ventricular end-diastolic pressure, whereas left ventricular peak systolic pressure, rate of pressure rise and decay were reduced. The hypertrophic response was modest, since statistically significant increases of 11% and 23% in left and right ventricular weights were measured. Radioligand binding documented an 18% and a 38% statistically significant reduction in alpha 1-adrenoreceptor density of the left and right myocardium, respectively. ADP ribosylation of the 41-kDa substrate by pertussis toxin showed a 16% significant decrease of this parameter in the left myocardium. Moreover, a 29% decrease in norepinephrine stimulated phosphoinositol turnover was seen in myocytes, and this change was statistically significant. The depressed generation of intracellular second messengers linked to the alpha 1-adrenoreceptors was found in conjunction with a 19% significant reduction in myocardial norepinephrine content. In conclusion, the long-term effects of coronary stenosis involve impaired transduction of adrenergic signals, which, in combination with constraints on myocardial growth, may participate in the onset of ventricular dysfunction in this model.


Intracellular signal transduction in four dimensions: from molecular design to physiology. Designed fluorescent indicators are the basis for a major new technique in cell physiology, the quantitative measurement and dynamic imaging of intracellular concentrations of important ions and messengers such as Ca2+, Na+, H+, and adenosine 3',5'-cyclic monophosphate. Molecular engineering has now produced indicators with quite good selectivity and sensitivity for these analytes. In many cases, these probes can be introduced into large populations of cells by means of membrane-permeant chemical derivatives, so that the plasma membrane need never be disrupted or physically breached at any point. Like many other optical microscopic techniques, fluorescent indicators are readily applied to study living cells and tissues, with an unparalleled combination of spatial and temporal resolution. They offer one of the few methods for continuous nondestructive monitoring of dynamic intracellular biochemistry and signal transduction in single cells or subregions of cells.


Functional localization of adenosine receptor-mediated pathways in the LLC-PK1 renal cell line. The functional localization of three adenosine receptor-mediated signal transduction pathways in the LLC-PK1 renal cell line was investigated. LLC-PK1 cells were grown on Millicell-CM filter inserts, which allow for the independent exposure of the apical or basolateral side of a confluent cell monolayer to hormones. Adenosine stimulated inositol phosphate turnover, inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (A1 receptor), and stimulation of cAMP accumulation (A2 receptor). Adenosine (10 microM) selectively applied to the basolateral side induced a significant (P < 0.05) increase in inositol phosphates, whereas apical exposure did not. The adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 microM), blocked the stimulation of inositol phosphate production in LLC-PK1 cells, provided support for an adenosine receptor-mediated event. When adenosine (30 nM) was selectively applied to the apical side, forskolin-stimulated cAMP levels were not significantly decreased (approximately 8%, P > 0.05). However, adenosine (30 nM) presented to the basolateral side produced a significant decrease (approximately 23%, P < 0.05) in forskolin-stimulated cAMP levels. A high dose (100 microM) of adenosine elicited a significant increase (P < 0.05) in cAMP levels when presented to either the apical or the basolateral cell surface. Adenosine (100 microM) applied to the apical side elicited significantly higher cAMP levels (P < 0.05) than the same dose applied basolaterally. LLC-PK1 cells grown on permeable supports exhibit a polarity of functional responses following activation by adenosine. These data support a topographic separation of the multiple adenosine signaling systems in a renal epithelial cell line.


Converting-enzyme inhibitors increase converting-enzyme mRNA and activity in endothelial cells. Exposure to angiotensin-converting-enzyme (ACE) inhibitors has been associated with increased ACE activity in vivo and in vitro. In the current study, we examined the effects of the active site-directed ACE inhibitors lisinopril and captopril on ACE gene expression and activity in cultured porcine pulmonary artery endothelial cells. Exposure of endothelial cells to both lisinopril and captopril was associated with increased ACE mRNA levels and concomitant increases in ACE activity. These effects were both concentration and time dependent. ACE mRNA levels began to increase within 30 min of ACE inhibitor exposure and showed an early peak at 2 h and a higher, delayed peak at 48 h. ACE activity peaked at 24 h. Both ACE mRNA levels and activity were highest during incubation with 100 microM inhibitor. Nuclear runoff assays indicated that 48 h of exposure to 100 microM of either captopril or lisinopril increased ACE gene transcription approximately threefold relative to a tubulin control, a level comparable to the increases in ACE mRNA levels and activity observed during ACE inhibitor exposure. These findings support the hypothesis that ACE gene expression endothelial cells is stimulated by active site-directed ACE inhibitors in vitro. This provides a molecular mechanism for the observation that plasma and tissue ACE activity in vivo is increased during chronic exposure to ACE inhibitors.


Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule. Because the presence of the angiotensin II (ANG II)-dependent phosphoinositide hydrolysis has been questioned from studies in proximal cells in culture, we looked for this transduction pathway in suspension of freshly isolated rat proximal tubule fragments. ANG II-receptor activation induced a prompt (within 15 s) and sustained increase in [3H]inositol phosphates (IPs; inositol trisphosphate, inositol bisphosphate, and inositol monophosphate). In fura-2-loaded tubules, it elicited a rapid and biphasic rise in cytosolic free calcium ([Ca2+]i) with an early peak (within 15 s) followed by a plateau. The peak was maintained in the absence of extracellular calcium. ANG II-induced inositol trisphosphate and [Ca2+]i rises showed a similar dose dependency, with a 50% effective concentration (EC50) of 2.9 and 5.5 nM, respectively. We checked that ANG II inhibited basal (EC50 4.4 nM) and parathyroid hormone- and forskolin-stimulated cAMP production, the latter effect being inhibited by pertussis toxin pretreatment. The effects of ANG II on IPs and [Ca2+]i were inhibited by the ANG II receptor subtype 1 (AT1) antagonist losartan and not by the ANG II receptor subtype 2 (AT2) antagonists PD 123177 and PD 123319. The effect of ANG II on forskolin-stimulated cAMP was inhibited by losartan and not by PD 123319. In agreement with these results, specific binding of 125I-[Sar1,Ile8]ANG II was markedly inhibited by losartan, whereas PD 123319 had no effect. These results demonstrate that AT1 receptor subtypes are present in intact rat proximal tubule cells and are coupled to both IPs-Ca2+ and cAMP signaling pathways. No evidence for AT2 receptor subtype is found.


Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation. The effect of thyroid status on the expression of cytochrome c oxidase (CYTOX) and the activities of citrate synthase (CS) and phosphofructokinase (PFK) were examined in chronically stimulated (10 Hz; 35 days) and contralateral, nonstimulated rat tibialis anterior muscle of hypothyroid, hyperthyroid, and euthyroid animals. Stimulation increased CYTOX activity by 2.7-, 3.2-, and 4.9-fold in hyperthyroid, euthyroid, and hypothyroid animals, respectively, to similar absolute values. CS displayed similar increases. Stimulation reduced PFK activity in hypothyroid and euthyroid animals to 45% and 60% of control values. This effect was abolished with hyperthyroidism. Thus stimulation and thyroid hormone act antagonistically on PFK activity. Stimulation increased CYTOX subunit III (mitochondrially encoded) mRNA by 2.5- and 2.9-fold in hyperthyroid and euthyroid animals. Similar increases were observed in the nuclear-encoded mRNAs of CYTOX subunit VIc in euthyroid muscle. In hyperthyroid and euthyroid conditions, the mRNA changes paralleled the increases in enzyme activity. In hypothyroid muscle, the increase in mRNA was less for subunit VIc than III, suggesting that hypothyroidism upsets the coordinate expression of nuclear and mitochondrial genes. Further, the increases in CYTOX activity exceeded that of both subunit mRNAs in hypothyroid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Increase of apamin receptors in skeletal muscle induced by colchicine: possible role in myotonia. We have shown an increase of apamin receptors in rat skeletal muscle membranes following the application of colchicine to the sciatic nerve. 125I-apamin binding to partially purified membrane fractions was observed since day 4, reached a maximum around days 6-15, and was negligible at day 35 after the application of colchicine. Control muscles (nerves treated with buffer solution) showed low binding values (11 fmol/mg protein). Maximal 125I-apamin binding values to partially purified muscle membranes of colchicine-treated rats (42 fmol/mg protein) were lower than those obtained in denervated muscle (95 fmol/mg protein). The affinity binding constant values were 37 (colchicine) and 95 pM (denervation). No signs of muscle denervation were observed on histological examination of the nerve submitted to colchicine treatment nor in the muscles innervated by it. Muscle tension developed by indirect stimulation was the same as in controls. We here show also that partially purified membranes of normal untreated muscles have measurable amounts of 125I-apamin binding (13 fmol/mg protein), similar to those obtained in control muscles. Electromyographic recordings of the muscles after colchicine treatment of the nerve showed abnormal repetitive electrical discharges, similar to myotonic discharges, that were present with a similar temporal course as the increase in apamin receptors. The myotonic-like discharges were suppressed by the topical application of apamin to the muscle, whereas the toxin had no effect on anthracene-9-carbolytic acid-induced myotonia. Our results suggest that a neurotrophic factor that travels by axonal flow is involved in the regulation of the expression of apamin receptors in skeletal muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)


Adrenalectomy reduces alpha 1 and not beta 1 Na(+)-K(+)-ATPase mRNA expression in rat distal nephron. The abundance of mRNA of alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-isoforms of Na(+)-K(+)-ATPase was examined in several renal structures of normal and adrenalectomized (ADX) rats. In situ hybridization with 35S-labeled cRNA probes was performed on kidney sections from adult rats. The number of silver grains per unit surface area was quantified over cells of the glomerulus, proximal convoluted tubules (PCT), early distal tubules (EDT), and cortical collecting ducts (CCD). In normal rat kidney, alpha 1- and beta 1-mRNA was detected in PCT, EDT, and CCD, with the following range of magnitude: EDT > CCD > PCT > glomerulus. The amount of alpha 1- and beta 1-mRNA was equivalent. A large abundance of these two mRNA species was also found in the medullary thick ascending limb of the loop of Henle. Expression of alpha 2, alpha 3, and beta 2 was very low and evenly distributed over any cell type. In ADX, a significant decrease in alpha 1-mRNA (30%) was observed in EDT and CCD, with no change in PCT. beta 1-mRNA abundance was unaffected by adrenalectomy. These results indicate that 1) in the rat kidney alpha 1- and beta 1-mRNA are coexpressed at a similar level that varies along the renal tubule according to the cell type, 2) minute expression of alpha 2-, alpha 3-, and beta 2-mRNA is present in the kidney, and 3) corticosteroid depletion reduces the expression of alpha 1- and not beta 1-mRNA in the corticosteroid-sensitive tubular cells.


Permeation and inactivation by calcium and manganese of bovine adrenal chromaffin cell calcium channels. Stimulation of fura-2-loaded bovine chromaffin cells with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 10 microM) or depolarization with high [K+] (50 mM) accelerated the entry of both Ca2+ and Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Removal of extracellular Na+ prevented the effects of DMPP but did not modify the effects of K+, indicating that Na+ is necessary for coupling of Ca2+ entry to the nicotinic receptor activation and that the ionophore associated with it is functionally impermeable to divalent cations. DMPP- as well as K(+)-evoked Ca2+ and Mn2+ influx were blocked completely by Ni2+ but only partially by dihydropyridines, suggesting that, in addition to L-type Ca2+ channels, other Ca2+ entry pathways may be present. Inactivation of Ca2+ channels, followed by comparing the rates of Mn2+ uptake at different time periods after the addition of DMPP or high K+, did not happen in the absence of extracellular Ca2+. When 1 mM Ca2+ was present, a delayed inhibition (half time, 10-20 s) was observed, suggesting that it is not due to the entry of Ca2+ itself but to the increase of the cytoplasmic Ca2+ concentration ([Ca2+]i) that takes a few seconds to develop. The influx of Ca2+, estimated from the increase of [Ca2+]i, was also impaired in a time-dependent fashion by previous entry of Mn2+. Inactivation of Ca2+ entry was achieved at estimated mean intracellular Mn2+ concentrations as low as 10(-9) M.


Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. We used patch-clamp methods to examine the effects of depletion and readdition of aldosterone on single, highly selective, amiloride-blockable sodium channels in the A6 cell line. Single-channel characteristics changed little before 24 h of continuous aldosterone depletion, although there was some reduction in short-circuit current. Thereafter, apical sodium permeability, measured as product of channel number per patch and individual channel open probability (NPo), was reduced between five- and sevenfold, primarily due to a large decrease in channel mean open time. With about the same time course, short-circuit current also decreased approximately fivefold. Readdition of aldosterone to depleted cells produced an increase in NPo within 2 h, primarily through an increase in mean open time. After readdition, channel number per patch increased twofold compared with cells not hormone deprived, with a return to control levels between 24 and 48 h after continuous exposure. The increase in short-circuit current followed a similar time course. The primary effect of aldosterone appears to be modulation of the open time of channels continuously present in the apical membrane, rather than promotion of the appearance or disappearance of channels from the membrane. In particular, it cannot be demonstrated statistically that aldosterone removal reduces the number of channels per patch, and there may actually be up to a twofold increase after a long period of aldosterone depletion.


Isolation of a chloride channel-enriched membrane fraction from tracheal and renal epithelia. A membrane fraction, enriched in Cl- channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The fraction (MPS) shows a 37-fold enrichment of Cl- channels over crude tracheal homogenates by net Cl- flux measurements. Alkaline phosphatase and Na(+)-K(+)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. Marker enzyme analysis for major subcellular membranes also proved negative. The MPS fraction exhibits a protein profile unlike that of other membrane fractions, with major proteins of 200 and 42 kDa, proteins of 30-35 kDa, and lesser amounts of other proteins. Reconstitution of MPS fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (H. H. Valdivia, W. P. Dubinsky, and R. Coronado. Science Wash. DC 242: 1441-1444, 1988). In addition, the voltage dependence, selectivity sequence of Cl- > Br- > or = I-, and inhibition by low concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid correspond to those of the predominant apical membrane channel. Thus, although the MPS appear to be of subcellular origin, they may be functionally related to an apical membrane Cl- permeability.


Increased alpha 1-adrenoceptor density in brown adipose tissue indicates recruitment drive in hypothyroid rats. The effects of hypothyroidism on whole body thermogenesis, brown adipose tissue recruitment state, and alpha 1-adrenergic receptor density were investigated. Treatment of rats with methimazole for 4-5 wk led, as expected, to reduction of growth and resting metabolic rate. The thermogenic response to norepinephrine injection was practically abolished. Generally, only small effects of hypothyroidism on brown adipose tissue were observed: total protein content, mitochondrial GDP binding capacity, and total content of the uncoupling protein thermogenin were not altered. The density of beta-adrenergic receptors (estimated with [3H]CGP-12177 as a ligand) was also unchanged. However, the density of alpha 1-adrenergic receptors (estimated with [3H]prazosin) was markedly increased; in other physiological conditions, such an increase has been associated with an increased degree of recruitment of the tissue. These data indicate that brown adipose tissue in the subthermoneutral hypothyroid animal, probably due to homeostatic mechanisms, is exposed to an increased sympathetic stimulation, leading to an increased alpha 1-adrenoceptor density. However, other features of recruitment are only poorly induced, probably due to attenuation of the beta-adrenergic signaling mechanism. The increased alpha 1-adrenergic receptor density may be responsible for certain altered features of brown adipose tissue in hypothyroid animals, such as peroxisomal recruitment and perhaps also for maintenance of the thermogenin content. The results also indicate that the increased alpha 1-adrenergic density generally seen in recruitment would not result from chronic beta-adrenergic stimulation of the tissue but may be controlled via another regulatory pathway, e.g., via the alpha 1-adrenergic pathway itself.


Sepsis-induced insulin resistance in rats is mediated by a beta-adrenergic mechanism. Bacterial infection decreases insulin-mediated glucose uptake (IMGU) by skeletal muscle and produces whole body insulin resistance. Because circulating catecholamines are elevated by the septic insult, the present study was performed to determine the potential role of the beta-adrenergic system in eliciting these changes. Before induction of sepsis, an infusion containing saline, propranolol, or atenolol was started and continued throughout the experimental protocol. Sepsis increased the basal rate of glucose production and utilization and impaired IMGU by peripheral tissues. The peripheral insulin resistance in septic rats was manifested by an increase in the dose producing 50% of maximal response and a decrease in the maximal responsiveness. Infusion of propranolol, a nonselective beta-adrenergic antagonist, attenuated the sepsis-induced increase in basal glucose turnover by 70% and completely prevented the decrease in IMGU by the whole body. In contrast, atenolol, a selective beta 1-antagonist, did not alter the glucose metabolic response to infection. Under basal conditions, propranolol prevented or attenuated the increase in glucose uptake by the gastrocnemius, diaphragm, skin, liver, lung, spleen, and ileum normally observed in septic rats. In addition, propranolol prevented the decrease in IMGU by various muscles and skin in septic animals. These results suggest that adrenergic stimulation, probably mediated by a beta 2-adrenergic mechanism, is partially responsible for the sepsis-induced increases in basal whole body glucose turnover and plays a prominent role in the development of peripheral insulin resistance in this condition.


Pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach. To evaluate directly the actions of cellular mediators on pepsinogen secretion, a nearly homogeneous population of dispersed chief cells was permeabilized using the bacterial toxin streptolysin O (30 IU/ml for 10 min). This resulted in the release of > 60% of cellular lactate dehydrogenase activity, whereas cellular pepsinogen levels were not altered. Examination of permeabilized cells using light and electron microscopy revealed preservation of cellular polarity and organelles. Pepsinogen secretion from permeabilized chief cells could be stimulated with increasing concentrations of calcium (300 nM to 10 microM), adenosine 3',5'-cyclic monophosphate (cAMP; 1 microM to 1 mM), phorbol 12-myristate 13-acetate (10 nM to 1 microM), or by addition of carbamylcholine (0.1 mM) to the incubation solution. In the absence of calcium [0.4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], activators of protein kinase C stimulated a two- to threefold increase in pepsinogen secretion, and potentiation of secretion was observed when these agents were combined with 0.3 and 1.0 microM calcium. In contrast to the effects of activators of protein kinase C, cAMP-induced pepsinogen secretion was observed only with calcium concentrations > or = 100 nM. Combinations of increasing concentrations of cAMP and calcium resulted in an additive secretory response. This study indicates the utility of streptolysin O-permeabilized chief cells for the study of signal transduction mechanisms that mediate pepsinogen secretion.


Rat intestinal angiotensin-converting enzyme: purification, properties, expression, and function. Angiotensin-converting enzyme [ACE (peptidyl-dipeptidase A, EC 3.4.15.1)] was purified from a total cell membrane fraction of rat intestinal mucosa. A 4,500-fold purification was achieved after affinity chromatography with lisinopril-Sepharose and gel filtration. The final preparation was judged to be homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 160,000. The purified protein is a glycoenzyme containing 12% N-linked carbohydrate. Purified ACE had a specific activity of 65 U/mg protein with benzoyl-Gly-His-Leu as substrate. A kinetic analysis showed that the enzyme had the maximal velocity with substrates containing proline at the COOH-terminal end. Inhibitor studies indicated that the enzyme is a metalloprotein. Along the proximal-distal axis of the small intestine, ACE activity is most predominant in the proximal to middle portions, decreasing toward the distal end. This pattern was also observed for ACE mRNA and protein, suggesting that ACE expression is controlled at the level of mRNA. Perfusion of benzoyl-Gly-His-Leu in vivo through a segment of intestinal jejunum demonstrated that ACE is an important intestinal dipeptidyl carboxypeptidase, participating in the digestion and assimilation of dietary peptides.


Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro. We sought to characterize the excitatory effect of thyrotropin-releasing hormone (TRH) in dorsal motor nucleus of the vagus (DMV) motoneurons by using the patch-clamp technique in rat brain stem slices. In our initial studies we used the cell-attached recording configuration using concentrations of TRH from 1 to 30 microM. Exposure of DMV motoneurons to TRH resulted in a concentration-related increase in spontaneously occurring action potential firing rate. This was observed in 63 of 85 DMV neurons (75%) tested and was unrelated to their location rostral or caudal to the obex. Invariably, desensitization occurred to the excitatory effect of TRH. Subsequent experiments using whole cell recordings in the current-clamp mode confirmed that TRH excites DMV neurons located both rostral and caudal to the obex. In the current-clamp configuration, TRH produced depolarization; i.e., 30 microM TRH elicited a depolarization of 8.7 +/- 3.2 mV (P < 0.05, n = 7). Studies using whole cell current recordings in voltage-clamp mode indicated that TRH in a concentration-dependent manner produces a small inward current that is associated with a decrease in the input resistance of -42.5 +/- 15.6 M omega (TRH 30 microM). TRH-induced inward current was also present under conditions of inhibition of synaptic transmission (i.e., in the presence of tetrodotoxin and cobalt). We also found that TRH reduced in a concentration-dependent manner both the fast transient A-type K+ current (IA) and the Ca(2+)-dependent afterhyperpolarizing current (IAHP). Using the extracellular recording technique in the cell-attached configuration, we investigated whether any part of TRH-induced increase in firing rate was due to an increase in the synaptic release of L-glutamate or acetylcholine. Prior exposure of DMV neurons to either kynurenic acid or to atropine did not antagonize any of the excitatory effect of TRH. Finally, we observed that addition of 30 microM TRH to the perfusing solution produced an increase in spontaneously occurring excitatory postsynaptic currents (EPSCs). This occurred without any change in the amplitude of EPSCs. These results indicated that TRH-induced increase in firing of DMV neurons is due to direct postsynaptic effects to activate an inward cationic current and to counteract IA and IAHP, as well as a presynaptic effect to increase the frequency of EPSCs.


Secretin stimulates bile ductular secretory activity through the cAMP system. Although convincing evidence has been obtained to support a ductular origin of secretin choleresis, the precise mechanism of the choleretic effect of the hormone is poorly understood. The present studies were carried out to 1) further clarify the anatomic site at which secretin stimulates bile flow and 2) establish the signal transduction system underlying this effect. To this end, parenchymal and nonparenchymal liver cells, the latter enriched in bile duct cells, were isolated from rats with ductular cell hyperplasia, and the effect of secretin on intracellular formation of both adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (IPs) was compared with that observed with glucagon and [Tyr10,13,Phe22,Trp25]secretin (SG-secretin). In the pancreas, secretin stimulates both messenger systems, while SG-secretin activates only the cAMP cascade. In isolated hepatocytes, both secretin and SG-secretin failed to increase formation of cAMP and IPs, which were instead activated by glucagon. In isolated bile duct cells, secretin induced formation of both cAMP and IPs, while SG-secretin stimulated solely the cAMP system, as in the pancreas. Glucagon did not stimulate either messenger system in this cell preparation. In vivo, both secretin and SG-secretin stimulated a bicarbonate-rich fluid in rats with bile ductular cell hyperplasia and in normal guinea pigs, which was demonstrated to originate at the distal biliary epithelium. These findings support the existing view that glucagon stimulates canalicular bile flow, while secretin increases secretory activity at the bile ductules and/or ducts. More importantly, they indicate that stimulation of ductular secretory activity by secretin is mediated by the cAMP system and does not involve the IP signal transduction pathway.


Maturation of inositol 1,4,5-trisphosphate receptor binding in rabbit tracheal smooth muscle. The mechanisms underlying maturational changes in agonist-mediated airway contractility remain to be identified. Since the signal transduction process coupled to airway contraction involves the Ca(2+)-mobilizing action of the second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], the present study examined 1) whether Ins(1,4,5)P3 binding to its intracellular receptor varies with age in rabbit tracheal smooth muscle (TSM), and 2) whether Ca2+ exerts a modulatory effect on Ins(1,4,5)P3 receptor binding that is age dependent. [3H]Ins(1,4,5)P3 binding was assayed in crude TSM membrane preparations isolated from 2-week-old and adult rabbits. Monophasic Scatchard plots were obtained, reflecting a single binding site, with Hill coefficients of 0.988-0.996. The mean +/- SE values for receptor density (Bmax) and binding affinity [i.e., the dissociation constant (Kd)] were similar in the adult and immature tissues, wherein Bmax = 211 +/- 6 238 +/- 60 fmol/mg protein, respectively; and Kd = 14.1 +/- 0.2 and 11.6 +/- 1.2 nM, respectively. Addition of Ca2+ (10(-8)-10(-3) M) significantly modulated Ins(1,4,5)P3 binding, with opposing maturational effects. In adult tissues, Ca2+ produced dose-dependent inhibition of Ins(1,4,5)P3 binding to 59.5% control, whereas Ins(1,4,5)P3 binding in response to Ca2+ was significantly enhanced in the 2-week-old tissues to 183% control. Collectively, these observations demonstrate that the inherent basal binding characteristics of the Ins(1,4,5)P3 receptor are similar in maturing TSM, but that Ca2+ exerts opposite modulatory actions on Ins(1,4,5)P3 receptor binding in immature and adult tissues.(ABSTRACT TRUNCATED AT 250 WORDS)


Impaired ability of prostaglandins to buffer renal vasoconstriction in genetically hypertensive rats. The purpose of this study was to gain insight into the mechanism(s) responsible for the exaggerated angiotensin II (ANG II)-induced renal vasoconstriction during the development of hypertension. In previous studies we observed that ANG II produces a twofold larger decrease in renal blood flow (RBF) in spontaneously hypertensive (SHR) compared with Wistar-Kyoto (WKY) rats before but not after cyclooxygenase inhibition. We suggested that this strain difference could be attributed to differences in renal prostaglandin (PG) levels and/or action. To evaluate these possibilities, measurements of RBF were made in 6-wk-old, anesthetized SHR and WKY pretreated with indomethacin. ANG II was injected intrarenally before and during continuous intrarenal infusion of a low dose of PGE2, viprostol (PGE2 analogue), PGI2, iloprost (PGI2 analogue), and bradykinin. In the control period ANG II reduced RBF by 50% in both strains. Infusion of PGs reduced the vasoconstrictor effect of ANG II in WKY, but had no effect in SHR. In contrast, infusion of bradykinin blunted the ANG II-induced vasoconstriction to a similar degree in both WKY and SHR. To investigate whether this lack of protection in SHR is due to strain differences in the number and/or affinity of renal receptors of PGs, radiolabeled ligand binding studies for PGE2 and PGI2 receptors were undertaken in glomeruli isolated from young WKY and SHR. Scatchard analysis revealed a single, high-affinity receptor site for PGE2 that was similar in both strains of rats. Both strains also exhibited a single, high-affinity PGI2 receptor site. No differences were observed in the PGE2 or PGI2 receptor number between WKY and SHR.(ABSTRACT TRUNCATED AT 250 WORDS)


Actions of lipoproteins in cultured human mesangial cells: modulation by mitogenic vasoconstrictors. Recent studies have suggested that hypercholesterolemia may aggravate glomerulosclerosis. Mesangial cells actively participate in this process. To elucidate mechanisms by which lipids act on human mesangial cells (HMC), we measured the receptor-specific uptake of apolipoprotein (Apo) B- and Apo B- and E-containing lipoproteins in the presence and absence of growth factors and studied the growth-related mechanisms in HMC after exposure to low-density lipoprotein (LDL). Human LDL and very low density beta-lipoprotein (beta-VLDL) isolated from cholesterol-fed rabbits were bound, internalized, and degraded by a receptor-specific mechanism (dissociation constants for degradation LDL 30.0 and for beta-VLDL 4.1 micrograms protein/ml medium). Maximal capacities were 30-50% of those of human fibroblasts. Acetylated and copper-oxidized LDL were not taken up specifically, suggesting no active scavenger-receptor activity. Preexposure to endothelin-1 (5 x 10(-7) M) and platelet-derived growth factor (PDGF A, B, 83 x 10(-12) M) for 16 or 15 h, respectively, doubled the uptake of LDL by HMC. In addition, PDGF synergized with LDL in stimulating DNA synthesis. Exposure of HMC to LDL resulted in a transient elevation of mRNA that encodes c-fos and c-jun, with a maximal effect seen after 30-60 min. In addition, PDGF A- and B-chain mRNAs were transiently elevated, peaking at 3 h in response to LDL (25 micrograms protein/ml medium) and continued to increase in a concentration-dependent manner (25-75 micrograms protein/ml medium). These data demonstrate that HMC take up lipoproteins via a receptor-specific mechanism with a high affinity for Apo E-containing lipoproteins which are often found in plasma of patients with renal disease. Vasoconstrictor and mitogenic peptides enhance lipoprotein receptor activity and have a synergistic effect on the mitogenic effect of LDL. LDL stimulates a number of growth-related genes. These data suggest that lipoproteins may play a critical role in mediating mesangial cell hypertrophy or proliferation, events intimately involved in the development of glomerulosclerosis.


Vasopressin resistance in potassium depletion: role of Na-K pump. Resistance to the hydrosmotic effects of vasopressin has been described in K depletion. It is not clear whether other effects of vasopressin, notably its effects on the Na-K pump in the collecting duct, are similarly affected. Adrenalectomized male Sprague-Dawley rats were allocated to either a normal K (NK) or low-K (LK) diet. Na-K pump activity (pmol.mm-1.h-1) in cortical collecting duct (CCD) and medullary collecting duct (MCD) was determined at 21 days after allocation to the dietary groups before and after exogenous vasopressin (0.1 U twice daily for 3 days). In animals on NK diet, vasopressin (AVP) led to a doubling of Na-K pump activity in the CCD from 502 +/- 47 to 1,144 +/- 41 pmol.mm-1.h-1 (P < 0.01). In K-depleted animals, which had a higher baseline Na-K pump activity, an increase was also observed from 1,056 +/- 97 to 1,239 +/- 65 pmol.mm-1.h-1 (P < 0.05), but this increase was quantitatively less, with the change being 183 vs. 642 pmol.mm-1.h-1 in K-replete rats. The findings in the MCD were similar; in rats on a NK diet, AVP led to a significant increase in Na-K pump activity from 498 +/- 29 to 830 +/- 28 pmol.mm-1.h-1 (P < 0.01). With K depletion, this directional change was preserved, increasing from 1,380 +/- 49 to 1,556 +/- 45 pmol.mm-1.h-1 (P < 0.05), but was quantitatively less than in K-replete rats, the change being 176 vs. 332 pmol.mm-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation. This study examines the ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8- to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis and acidosis using 15 mM NH4+ salt. The net recovery rate (dpHi/dt) from intracellular acidosis increases significantly between 10 and 12 days of age from 0.39 +/- 0.04 to 0.54 +/- 0.06 pH units/min (P < 0.05, n = 10 vs. 6). This increase can be completely accounted for by an increase in the rate of amiloride (100 microM)-inhibitable Na(+)-H+ exchange (0.29 +/- 0.04 vs. 0.42 +/- 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na(+)-H+ exchange increases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increased by Na+ depleting the cells (48 and 49%, respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS, 500 microM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosis is significantly lower in RPTC-10 than in RPTC-40 (0.16 +/- 0.02 vs. 0.28 +/- 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500 microM) inhibits the recovery by 27 +/- 8 and 26 +/- 9%, respectively, whereas amiloride has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)


Ouabainlike factor in Milan hypertensive rats. Ouabainlike factor (OLF) has been extracted from the hypothalamus and adrenals of the ox and rats of the Milan hypertensive strain (MHS) and their normotensive controls (MNS). OLF was identified by its ability to 1) inhibit ouabain-sensitive 86Rb uptake into human erythrocytes, 2) displace [3H]ouabain binding, and 3) inhibit purified dog kidney Na-K-adenosinetriphosphatase (ATPase). Rat and bovine OLF have similar characteristics. Those that are close to ouabain are 1) ligand conditions for maximal inhibitory activity, 2) high-performance liquid chromatography retention time, 3) reversibility of inhibitory activity on Na-K-ATPase, 4) reduced Na-K pump inhibitory activity by K, 5) high affinity for Na-K-ATPase, and 6) no activity on calcium ATPase. OLF does not resemble ouabain in the following characteristics: 1) the capacity of OLF to inhibit ouabain low-affinity Na-K-ATPase isoform is greater than that of ouabain and 2) the capacity of OLF to inhibit renal Na-K-ATPase isoforms is greater when the enzyme is obtained from adult rather than young rats. The yield of OLF is greater from MHS than MNS. These findings represent the first direct evidence that a higher amount of OLF is present in tissues from genetically hypertensive rats than from their inbred normotensive controls, maintained under the same dietary and environmental conditions. This further supports previous observations on the role of OLF in the pathogenesis of MHS hypertension.


Effects of CD-349 and 8-BrcGMP on isoproterenol-induced relaxation in rabbit aorta precontracted with endothelin-1. We investigated the effects of CD-349, a dihydropyridine derivative, on isoproterenol (Iso)-induced relaxation in rabbit aorta precontracted with endothelin-1 (ET-1). The Iso (10(-8)-10(-5) M)-induced relaxation responses in rabbit aorta precontracted with 1-2 x 10(-7) M ET-1 were augmented by pretreatment with CD-349 (10(-9)-10(-5) M) in a concentration-dependent manner. The effects of CD-349 on Iso-induced relaxation of the aortic strips precontracted with ET-1 were inhibited by treatment with methylene blue (10(-5) M) and oxyhemoglobin (10(-5) M), whereas they were augmented by treatment with N omega-nitro-L-arginine (10(-4) M). The Iso-induced relaxation responses were also augmented by pretreatment with 8-Br-guanosine 3',5'-cyclic monophosphate (cGMP) (3 x 10(-6)-3 x 10(-4) M) in a concentration-dependent manner. However, nifedipine (10(-5) M) and nicardipine (10(-5) M) had no effect on Iso-induced relaxation responses of the aortic strips precontracted with 10(-7) M ET-1. CD-349 also augmented forskolin (10(-8)-10(-5) M)-induced relaxation responses of rabbit aorta in a concentration-dependent manner. CD-349 (10(-7)-10(-5) M) increased the levels of cGMP but not of adenosine 3',5'-cyclic monophosphate (cAMP) in a concentration-dependent manner in rabbit aorta without endothelium. Both CD-349 (10(-5) M) and 8-BrcGMP (3 x 10(-5) M) augmented the Iso-induced elevations of cAMP in rabbit aorta without endothelium. These results indicate that CD-349 and 8-BrcGMP can augment Iso-induced relaxation responses by enhancing the accumulation of cAMP.


Local temperature modulates alpha 1- and alpha 2-adrenergic vasoconstriction in men. Previous work in the canine saphenous vein has shown that cooling augments alpha 2- but not alpha 1-mediated contractile responses and that warming produces the opposite effects. Here we sought to determine whether these results occur in the human finger, a cutaneous vascular bed. Healthy men received brachial artery infusions of phenylephrine and clonidine with and without yohimbine while sympathetic tone was reduced by hearing the legs. Finger blood flow was recorded by venous occlusion plethysmography from two fingers, one cooled and one uncooled, on each hand. Cooling augmented alpha 2-adrenergic vasoconstriction produced by clonidine; this effect was reduced by yohimbine, an alpha 2-adrenergic antagonist. In contrast, cooling abolished alpha 1-adrenergic vasoconstriction produced by phenylephrine, which was not affected by yohimbine. Further studies were conducted in which fingers were warmed rather than cooled. Warming augmented alpha 1- but reduced alpha 2-adrenergic vasoconstriction. Thus, in human fingers, cooling augments alpha 2- and suppresses alpha 1-adrenergic vasoconstriction, whereas warming produces the opposite effects.