Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Summary Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin >isoproterenol>calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin>vasopressin=PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin=calcitonin. Neither isoproterenol nor PTH had an effect.These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.     

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane-like material adjacent to the dish. Differentiated properties characteristic of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK-ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT-specific anti-keratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular epithelia in the kidney.     

Distribution of prostaglandin E2-sensitive adenylate cyclase along the rat nephron

To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E₂ (PGE₂) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE₂ and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE₂. PGE₂ stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10^?5 to 2.8 × 10^?5 M. PGE₂ was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE₂, at both high (2.8 × 10^?5 M) and low (2.8 × 10^?8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE₂-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE₂ on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.     

Lack of inhibition by atrial natriuretic factor on cyclic AMP levels in single nephron segments and the glomerulus

Recently an inhibitory effect of atrial natriuretic factor (ANF) on the adenylate cyclase system has been reported in vascular tissue. In seeking similar affects in renal tissue, we studied the effect of ANF on cyclic AMP levels in single nephron segments and in glomeruli from the rat. Individual nephron segments or glomeruli were incubated in the presence of a phosphodiesterase inhibitor, with or without parathyroid hormone (PTH) or arginine vasopressin (AVP) and varying concentrations of ANF at 37 degrees C for 2 min. The capacity for alpha 2-adrenoceptor inhibition of adenylate cyclase was demonstrated in the proximal convoluted tubule, cortical collecting tubule and in glomeruli. Nevertheless, ANF could not inhibit cAMP formation in any of these nephron segments nor in the glomerulus. Thus, unlike the vasculature, ANF has no inhibitory effect on cAMP formation in these renal tissues.     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.     

Cortical cell populations from rabbit kidney isolated by free-flow electrophoresis: characterization by measurement of hormone-sensitive adenylate cyclase

Free-flow electrophoresis allows the separation of different cell populations from a cell suspension isolated from rabbit kidney cortex after perfusion of the kidneys with a calcium-binder, followed by gentle mechanical treatment. After electrophoretic separation, analysis of the adenylate cyclase activities after stimulation by various hormones allows the precise determination of the origin of the cell populations with different electrophoretic mobilities. Adenylate cyclase from the slow-moving main cell population was only sensitive to parathyroid hormone. These cells had also high alkaline phosphatase content, further demonstrating their proximal origin. The various fast- moving cell populations had adenylate cyclase sensitive to isoproterenol and arginine vasopressin but were less sensitive to parathyroid hormone than the slow-moving cells. Their alkaline phosphatase content was also much lower. This indicates that these fast- moving cell populations originate from both the granulous segment of the distal tubule and from the collecting ducts. The adenylate cyclase activity and the cyclic AMP contents of isolated proximal cells maintained in culture medium were also investigated.     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Transforming growth factor beta enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast-like cells

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.     

Immunomagnetic separation,primary culture,and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney

Summary Renal cortical thick ascending limbs of Henle’s loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 × 10⁶ cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron. Experimental work reported here was supported by grants from the National Institutes of Health, Bethesda, MD GM34399, American Heart Association (grant-in-aid 88-0721), and the Hitchcock Foundation. J. H. Pizzonia was supported by a Ford Foundation Fellowship and this work constitutes partial fulfillment of the requirements for a doctoral degree at Dartmouth College. B. J. Bacskai was supported by a Pharmaceutical Manufacturers Association Foundation Advanced Predoctoral Fellowship. P. A. Friedman was an Established Investigator of the American Heart Association during the tenure of these studies.     

Maintenance of proximal and distal cell functions in SV40-transformed tubular cell lines derived from rabbit kidney cortex

This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum-free hormonally defined medium and display basic properties of proximal tubule cells including well-developed apical microvilli, strong expression of brush-border hydrolases, Na+-coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum-free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+-dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine vasopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1.     

Pertussis toxin prevents homologous desensitization of adenylate cyclase in cultured renal epithelial cells

The biochemical mechanisms of adenylate cyclase desensitization in arginine vasopressin-responsive epithelial cells remain unclear. Preincubation of cultured rabbit renal cortical collecting tubular cells with arginine vasopressin leads to a 30-100% decline in arginine vasopressin-stimulated adenylate cyclase activity. This loss of adenylate cyclase activity is time- and arginine vasopressin concentration-dependent. Preincubation with arginine vasopressin does not result in significant changes in basal, NaF-, forskolin-, isoproterenol- or cholera toxin-stimulated adenylate cyclase activity. Preincubation of cells with chlorophenylthio-cAMP, forskolin, and cholera toxin does not result in loss of arginine vasopressin-stimulated adenylate cyclase activity. Since products of cyclo-oxygenase inhibit arginine vasopressin action, cells were preincubated with indomethacin. Arginine vasopressin-induced adenylate cyclase desensitization is not reversed by indomethacin. By contrast, incubation with pertussis toxin prevents arginine vasopressin-induced adenylate cycle desensitization. These data demonstrate that arginine vasopressin induces homologous desensitization in membranes from cultured rabbit cortical collecting tubular cells and suggest that this desensitization is mediated, at least in part, by pertussis toxin substrate. These observations provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Calmodulin stimulation of adenylate cyclase in rat brain membranes does not require GTP

Calcium stimulates adenylate cyclase activity in rat cerebral cortical membranes with either ATP or AppNHp as substrate. In contrast, isoproterenol stimulates the cerebral cortical enzyme with ATP as substrate but not with AppNHp as substrate unless exogenous GTP is added. In rat striatal membranes, calcium or dopamine stimulate adenylate cyclase activity with ATP as substrate, but not with AppNHp as substrate. GTP restores the dopamine but not the calcium response. The inhibitory guanine nucleotide GDP-βS antagonizes dopamine and GppNHp stimulation of the brain adenylate cyclases, but not stimulation by calcium of either rat cerebral cortical or striatal enzymes. Results indicate that GTP is not requisite to calcium-calmodulin activation of adenylate cyclases in brain membranes. In addition, calcium-calmodulin cannot activate striatal adenylate cyclases with a nonphosphorylating nucleotide, AppNHp, as substrate.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

5'guanylyl-imidodiphosphate actions on adenylate cyclase in homogenates of rat cerebral cortex plus neuronal and capillary fractions

A variety of neurohumoral agents activate adenylate cyclase in homogenates of rat frontal cortex (norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 4-Me-histamine and prostaglandins E₁, E₂ and A₂). The enzyme in homogenates of isolated cortical neurons is likewise sensitive to norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 2-Me- and 4-Me-histamine, and prostaglandin F_2α. Capillary-enriched fractions from the cortex possess an enzyme that is activated by norepinephrine, isoproterenol and dopamine. Addition of 5′-guanylyl-imidodiphosphate (Gpp(NH)p) to the cortical homogenates and neuronal fractions resulted in enhanced enzyme responses to norepinephrine, isoproterenol, dopamine, 2-Me- and 4-Me-histamine and the prostaglandins E₁ and E₂. The actions of histamine and apomorphine were not increased by the GTP analog. The sensitivity of the catecholamine-induced adenylate cyclase activation in cortical capillaries was augmented by Gpp(NH)p. Thus various cellular types within the cerebral cortex may possess different receptor characteristics with respect to stimulation of adenylate cyclase by neurohormones.     

Modulation of parathyroid hormone-sensitive adenylate cyclase and arginine vasopressin-sensitive adenylate cyclase by calcium and GTP

Arginine vasopressin (AVP)- and parathyroid hormone (PTH)-sensitive adenylate cyclase were studied in the renal tissue of thyroparathyroidectomized dogs. The results indicate that AVP-sensitive adenylate cyclase activity was highest in the inner medulla followed by the middle medulla, outer medulla, and cortex, in declining order. In contrast, PTH-sensitive adenylate cyclase was absent in the inner medulla, and the highest stimulation was found in the cortex with lesser activity in outer and middle medulla. When 1 mm EGTA was included in the incubation mixture, the addition of both AVP- and PTH to the middle medullary homogenate resulted in additive responses suggesting two separate receptors for each hormone. This EGTA-induced additive effect was eliminated by the addition of calcium into the system, indicating that calcium concentration may be critical in modulating the interaction of AVP and PTH-sensitive adenylate cyclase. In contrast to some previous reports, a particulate fraction prepared from the middle medullary tissue was completely insensitive to either AVP or PTH. Hormonal sensitivity was restored by the addition of GTP or the supernatant.