GTP-independent stimulation of rabbit heart adenylate cyclase by isoproterenol at physiological ATP concentrations

Summary Isoproterenol increased the activity of the adenylate cyclase of rabbit heart sarcolemmal membranes in the absence of added GTP. ATP, the ATP-regenerating system, and the sarcolemmal membrane preparation were eliminated as possible sources of contaminating GTP. Isoproterenol-stimulation increased as ATP was raised. At 0.5 mM ATP, isoproterenol increased activity by 19% whereas at 5 mM ATP isoproterenol increased activity by 121%. There was no change in basal activity between 0.5 and 5 mM ATP. Stimulation by Gpp(NH)p and NaF increased slightly between 0.5 and 5 mM ATP; stimulation by KCl was unaffected. GTP does not activate cyclase d. GTP does not activate cyclase to the same extent as Gpp(NH)p even though the two act at the same site on Ns (the stimulatory guanine nucleotide-binding protein). GTP decreased cyclase activation by Gpp(NH)p in a concentration-dependent fashion when the two were added to the assay simultaneously. Increasing ATP from 0.5 to 5 mM did not reduce activation by Gpp(NH)p when both were added simultaneously to the assay. This suggests that ATP does not interact with the same site as Gpp(NH)p. ATPS, an analogue of ATP which irreversibly thiophosphorylates proteins, did not irreversibly support activation by isoproterenol. The effect of ATP in supporting isoproterenol stimulation is not, therefore, thought to be due to phosphorylation of a protein.     

Bile acid stimulation of colonic adenylate cyclase and secretion in the rabbit

In vitro studies have suggested that the cyclic AMP system may be the mediator of bile acid-induced colonic secretion. The aims of our experiments were to quantify thein vivo effect of various doses of deoxycholic acid (DCA) on adenylate cyclase activity (AC) and net secretion in the rabbit colon. AC increased significantly (P<0.01) with each increasing concentration of DCA; at 8 mM the activity was 126±6 pmoles cAMP/mg protein/min, or 4 times the control. Phosphodiesterase activity (PDE) was affected and significantly decreased (P<0.01) only by 8 mM DCA. The volume of luminal fluid increased significantly (P<0.01) as a bicarbonate-rich solution with 4, 6, and 8 mM DCA in graded fashion. In conclusion, stimulation of a colonic mucosal cAMP system is strongly implicated as mediating DCA-induced colonic secretion.Supported in part by NIH grant AM 15631.     

Inhibition by propranolol of bile acid stimulation of rabbit colonic adenylate cyclase in vitro.

Bile acids, especially unconjugated deoxycholic acid, cause diarrhea by inducing colonic mucosal secretion of water and electrolytes. This effect has been shown to be mediated by adenylate cyclase (AC). Propranolol, a beta-adrenergic blocking agent which inhibits AC, may then prevent this action of bile acids on colonic mucosa. The aim of this study was to determine the effect of bile acids, catecholamines, and propranolol on AC activity in colonic mucosa. The in vitro effects of deoxycholic acid, taurocholic acid, NaF, epinephrine, norepinephrine, and propranolol on AC in rabbit colonic mucosa were determined. NaF, 10(-4) M, increased AC activity to 220% of control (P less than 0.01). Deoxycholic acid, 10(-4) M, increased AC activity to 178% of control (P less than 0.01). Lesser but significant (P less than 0.01) stimulation of AC occurred at both higher and lower concentrations of deoxycholic acid, with no effect at 10(-10) M. Taurocholic acid, 10(-4) M, and epinephrine and norepinephrine, 10(-2) M, 10(-4) M, 10(-6) M, and 10(-8) M, had no effect on AC. Propranolol, 10(-6) M, caused a 60% decrease (P less than 0.01) in the stimulated AC activity induced by 10(-4) M deoxycholic acid. Propranolol, 10(-4) M, decreased basal AC by 30% (P less than 0.01). In conclusion: (1) Deoxycholic acid, but not taurocholic acid, epinephrine, or norepinephrine, stimulates colonic AC activity. (2) Propranolol inhibited this deoxycholic acid stimulation of AC. (3) Catecholamines are not intermediaries in this action of propranolol on colonic mucosal AC activity.     

Atrial natriuretic factor does not affect basal, forskolin- and CRF-stimulated adenylate cyclase activity, cAMP formation or ACTH secretion, but does stimulate cGMP synthesis in anterior pituitary

The report that ANF inhibits basal and CRF-stimulated adenylate cyclase activity in anterior pituitary homogenates suggested that the atrial peptide could inhibit ACTH secretion. This possibility was investigated in the ACTH-secreting AtT-20 mouse pituitary tumor cell line as well as homogenates or primary cell cultures from rat anterior hypophysis. ANF (up to 5 X 10(-7) M) was found to be completely ineffective in stimulating basal, CRF- and/or forskolin-stimulated adenylate cyclase activity, cAMP accumulation and ACTH secretion. Similarly, ANF had no effect on spontaneous or GRF-induced GH release from cells in primary culture. ANF receptors, however, are present in AtT-20 cells and anterior pituitary cells as evidenced by the ability of the peptide to stimulate intracellular cGMP accumulation. The data, therefore, suggests that ANF does not have a negative modulatory action on the secretory function of anterior pituitary. The role of cGMP in any other action(s) of ANF remains unknown.     

Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP.

The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.     

Effects of calcitonin gene-related peptide (CGRP) and isoproterenol on the contractility and adenylate cyclase activity in the rat heart

The occurrence of calcitonin gene-related peptide (CGRP) has been predicted by the study of the calcitonin gene of rats [1]. Recently CGRP was isolated from porcine spinal cord [3], and thus, CGRP is assumed to be a neuropeptide. It has been shown that CGRP exhibits potent positive chronotropic and inotropic effects on the atrial muscles of rats and guinea-pigs [5, 9, 11] . We have previously demonstrated the presence of nonadrenergic noncholinergic (NANC) nerves in the atria of rats and guinea-pigs [10] and have suggested that CGRP is a neurotransmitter of the NANC nerves in the guinea-pig atria [9, 11]. It is supposed that the positive inotropic response mediated by the beta-adrenoceptor results from a stimulation of the adenylate cyclase (AC) activity and subsequent elevation of cyclic AMP (cAMP) content. In the present study, the effects of calcitonin gene-related peptide (CGRP) and isoproterenol (ISO) on the myocardial contractility and adenylate cyclase (AC) activity were compared in cardiac muscles of the rat. In atrial muscles, CGRP exerted a positive inotropic effect and stimulated the AC activity at the similar dose range. On the contrary, the concentrations of ISO needed to stimulate the AC activity were nearly 10-fold higher than those needed for eliciting positive inotropic responses. In ventricular muscles, CGRP produced neither a positive inotropic responses nor a stimulation of AC activity, while ISO induced both responses. Inasmuch as CGRP-like immunoreactive (CGRP-I) nerves were found to be present in the atria but rarely in the ventricles, CGRP was assumed to be physiologically important in the atria rather than in the ventricles.     

Gonadotropin-mediated desensitization in a murine Leydig tumor cell line does not alter the regulatory and catalytic components of adenylate cyclase

MLTC-1 cells, derived from a murine Leydig tumor, contain a gonadotropin-responsive adenylate cyclase that became desensitized to hCG. Prior exposure to hCG reduced the ability of MLTC-1 cells to accumulate cAMP by approximately 50%, but caused only a small reduction in hCG receptor number. Membranes isolated from desensitized cells showed a similar reduction in hCG-stimulated adenylate cyclase activity. Desensitization was time, temperature, and dose dependent. Elevating intracellular cAMP levels by incubating the cells with (Bu)2cAMP or cholera toxin failed to cause desensitization. Desensitization did not depend on protein synthesis. Desensitization caused no change in the dose response of adenylate cyclase to hCG or GTP. hCG receptor affinity for hCG was not affected by desensitization or guanine nucleotides. The stimulatory regulatory component of adenylate cyclase (Ns) from MLTC-1 cells was used to reconstitute S49 cyc- membranes, which lack Ns. Ns from control and desensitized MLTC-1 cells were equally effective in reconstitution of the beta-adrenergic-sensitive adenylate cyclase of cyc-. beta-Adrenergic receptors from cyc- membranes were also transferred to MLTC-1 membranes by fusion with polyethylene glycol to produce a beta-adrenergic-responsive adenylate cyclase. Isoproterenol-stimulated activity was similar, regardless of whether membranes from control or desensitized MLTC-1 cells were used. We conclude that neither Ns nor the catalytic subunit of the adenylate cyclase in MLTC-1 cells is the site of lesion in desensitization. Most likely, the hCG receptor itself may be affected when MLTC-1 cells are desensitized by hCG.     

Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.

Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte ghosts and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5- to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.     

The hormonal control of adenylate cyclase in rabbit myometrium: in vitro inhibition by adenosine and lack of effect of progesterone

The mechanism by which progesterone modifies uterine smooth muscle cell contraction is still unknown. We investigated the biochemical basis of progesterone effects on myometrium of estradiol-primed rabbits. Progesterone did not affect adenylate cyclase basal activity and displayed no interaction with stimulators of myometrium adenylate cyclase (NaF, guanylyl nucleotides, beta-adrenoreceptor agonists, prostaglandins, forskolin) or with adenosine. On the other hand, adenosine inhibited myometrial adenylate cyclase, which correlates with its contracting properties in vivo; this inhibition is mediated through interaction at 'P sites'. We conclude that whilst adenosine inhibits myometrial adenylate cyclase by acting at 'P sites', progesterone does not interact directly with adenylate cyclase to regulate myometrial contractility.     

c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart

We examined the myogenic response to infarction in neonatal and adult mice to determine the role of c-kit(+) cardiovascular precursor cells (CPC) that are known to be present in early heart development. Infarction of postnatal day 1-3 c-kit(BAC)-EGFP mouse hearts induced the localized expansion of (c-kit)EGFP(+) cells within the infarct, expression of the c-kit and Nkx2.5 mRNA, myogenesis, and partial regeneration of the infarction, with (c-kit)EGFP(+) cells adopting myogenic and vascular fates. Conversely, infarction of adult mice resulted in a modest induction of (c-kit)EGFP(+) cells within the infarct, which did not express Nkx2.5 or undergo myogenic differentiation, but adopted a vascular fate within the infarction, indicating a lack of authentic CPC. Explantation of infarcted neonatal and adult heart tissue to scid mice, and adoptive transfer of labeled bone marrow, confirmed the cardiac source of myogenic (neonate) and angiogenic (neonate and adult) cells. FACS-purified (c-kit)EGFP(+)/(αMHC)mCherry(-) (noncardiac) cells from microdissected infarcts within 6 h of infarction underwent cardiac differentiation, forming spontaneously beating myocytes in vitro; cre/LoxP fate mapping identified a noncardiac population of (c-kit)EGFP(+) myocytes within infarctions, indicating that the induction of undifferentiated precursors contributes to localized myogenesis. Thus, adult postinfarct myogenic failure is likely not due to a context-dependent restriction of precursor differentiation, and c-kit induction following injury of the adult heart does not define precursor status.     

alpha1A- but not alpha1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism

OBJECTIVE: Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent ischemic injury. alpha1-Adrenergic receptors (ARs) have been implicated in this process. However, the lack of sufficiently selective antagonists has made it difficult to determine which alpha1-AR subtype protects the heart from ischemic injury. The goal of this study was to identify the alpha1-AR subtype that is involved in ischemic preconditioning. METHODS: We developed transgenic mice that express constitutively active mutant (CAM) forms of the alpha1A-AR or the alpha1B-AR regulated by their endogenous promoters. Hearts isolated from transgenic and non-transgenic mice were perfused by the Langendorff method using an ischemic preconditioning perfusion protocol or a non-preconditioning perfusion protocol prior to 30-min ischemia and 40-min reperfusion. Contractile function was continuously monitored through an intraventricular balloon. RESULTS: The contractile function of non-transgenic hearts perfused according to the ischemic preconditioning protocol completely recovered from 30-min ischemia. However, non-transgenic hearts perfused according to the non-preconditioning protocol recovered only 60% of their contractile function. The contractile function of CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, completely recovered from 30-min ischemia even though they were perfused according to the non-preconditioning protocol. Thus, CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, were inherently preconditioned against ischemic injury. Staurosporine, but not chelerythrine, completely reversed the preconditioning effect of CAM alpha1A-ARs. CONCLUSIONS: These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.     

The effects of trimetoquinol on the intact rabbit heart and myocardial adenylate cyclase activity: evidence for spare myocardial beta receptors

We have compared and contrasted the actions of (-)isoproterenol and (+/-) trimetoquinol on rabbit heart preparations. In the presence of either GTP or Gpp[NH]p (guanosine-5'-(beta, gamma imino) triphosphate), trimetoquinol displayed partial agonist activity in stimulating adenylate cyclase activity in a particulate rabbit heart preparation. Trimetoquinol enhanced adenylate cyclase activity 20% or 65% of the maximum obtainable by isoproterenol in the presence of GTP or Gpp[NH]p respectively. In the presence of GTP, concentrations of catecholamines required to enhance cyclase activity 15% of the maximum obtainable with isoproterenol (EC15) were 2.0 X 10(-7) M and 5.5 X 10(-8) M for trimetoquinol and isoproterenol, respectively. In the presence of Gpp[NH]p EC30 values were 2.0 X 10(-7) and 3.5 X 10(-8) M for trimetoquinol and isoproterenol respectively. Trimetoquinol also displayed partial agonist activity for the ability to increase cAMP levels in the isolated perfused rabbit heart. By contrast trimetoquinol was equieffective to isoproterenol at increasing tension development and rate of contraction of the isolated perfused heart. Concentrations of catecholamines required to increase tension and rate of contraction 50% of the maximum obtainable with isoproterenol were 1.5 X 10(-7) M and 1.7 X 10(-8) M for trimetoquinol and isoproterenol, respectively. These data show that only a partial stimulation of adenylate cyclase activity and cAMP levels by trimetoquinol is sufficient to produce maximal changes in mechanical activity of the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epinephrine stimulation of fat cell adenylate cyclase: regulation by guanosine-5'-triphosphate and magnesium ion

Guanosine-5′-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCl₂, GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 μM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity.GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent K_M for ATP and the V_max of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine.     

Decreased stimulation of adenylate cyclase by growth hormone-releasing factor in the anterior pituitary of old rats

Adenylate cyclase activity was studied in anterior pituitary homogenates from young adult (6 months) and old (20 and 24 months) male rats. Basal, NaF-, GTP-, 5'-guanylimidodiphosphate (Gpp(NH)p)- and forskolin-stimulated activities were similar in the three groups, implying that the stimulatory guanyl nucleotide regulatory binding site (NS) and the catalytical unit were unaffected by aging. Vasoactive intestinal peptide (VIP)-stimulated adenylate cyclase activity was also identical in the three groups. The efficacy (i.e., the maximum effect) of growth hormone-releasing factor [GRF(1-29)-NH2] on adenylate activity was reduced by 45-49% in old and senescent rats with no change in peptide potency (i.e., the concentration required for half-maximal enzyme stimulation). These results suggest that aging induced a selective loss of functional GRF receptors but influenced neither the coupling between receptors, NS and the catalytic unit nor the efficacy of the catalytic unit per se.     

Acute ingestion of red wine by men activates platelets but does not influence endothelial markers: no effect of white wine.

Long-term moderate alcohol use is associated with a better cardiovascular risk profile than total abstinence, although the short-term effect of a bolus of alcohol is unclear. The hypothesis tested in this study was that an acute bolus of alcohol would adversely affect the endothelium and platelets. Blood was taken before and 4 h after the ingestion of red or white wine by nine volunteers per group, and by 11 control water-only drinkers at the same time points. Plasma was obtained and markers of platelet activity (beta-thromboglobulin and soluble P selectin) and endothelial cell function (von Willebrand factor and soluble thrombomodulin) measured by enzyme-linked immunosorbent assay. The only marker to change significantly was beta-thromboglobulin, which increased from a median of 10 ng/ml (interquartile range, 8.5-15) before drinking red wine to 16 ng/ml (interquartile range, 14-20) 4 h later (P = 0.0067). We conclude that an acute bolus of red wine, but not white wine, activates platelets but has no substantial effect on the endothelium.     

Regional differences of adenylate cyclase stimulation by calcitonin and calcitonin gene-related peptide in the human kidney

Calcitonin (CT) gene-related peptide (CGRP)-like immunoreactivity was detected in both the cortex and medullo-papillary portion of human kidneys. The two forms of human CGRP as well as rat CGRP were capable of stimulating renal cortical adenylate cyclase activity in a concentration-related manner, with a half-maximally effective concentration (EC50) similar to that of human CT and approximately 100-1000 times higher than that of salmon CT. However, in the medullo-papillary portion, in which both salmon CT and human CT were inactive, the two forms of human and rat CGRP increased adenylate cyclase activity by 100%, with EC50 values ranging from 36 nmol/L to 1 mumol/L. In cortical membrane preparations the effect of CGRP was additive to that of salmon CT. We concluded that regional differences exist in the effect of CT and CGRP in human renal tissue and that in the medullo-papillary portion and possibly in the cortex, CGRP stimulates adenylate cyclase activity through a CT-independent mechanism.     

Reversal of repolarization gradient does not reverse the chirality of shock-induced reentry in the rabbit heart

INTRODUCTION: Two hypotheses have been proposed to explain the mechanisms of vulnerability and related failure of defibrillation therapy: the cross-field-induced critical point hypothesis and the virtual electrode-induced phase singularity hypothesis. These two hypotheses predict the opposite effect of preshock repolarization on the chirality (direction of rotation) of shock-induced reentry. The former suggests its reversal upon reversal of repolarization, whereas the latter suggests its preservation. The aim of this study was to determine, by reversing the repolarization sequence, which of the mechanisms is responsible for internal shock-induced arrhythmia in the Langendorff-perfused rabbit heart. METHODS AND RESULTS: We used high-resolution optical mapping to assess the chirality of postshock reentry in 11 hearts. Hearts were paced at a coupling interval of 300 msec at various sites around the field of view (13.5 x 13.5 to 16.5 x 16.5 mm). Cathodal monophasic implantable cardioverter defibrillator shocks (-100 V, 8 msec) were applied during the T wave from a 10-mm coil electrode placed into the right ventricular cavity. We used 3.5 +/- 0.8 different pacing sites per heart. Change in direction of repolarization did not result in change of chirality. Chirality was constant in all 11 hearts despite the complete reversal of activation and repolarization patterns. However, the position of resulting vortices depended on transmembrane polarization gradient inverted delta Vm and amplitude of negative polarization Vm (deexcitation). Stronger gradients and deexcitation produced earlier epicardial break excitation (P = 0.04 and P < 0.0001, respectively). CONCLUSION: Virtual electrode-induced phase singularity mechanism underlies internal shock-induced arrhythmia in this model.     

Cardiovascular and adenylate cyclase stimulating effects of colforsin daropate, a water-soluble forskolin derivative, compared with those of isoproterenol, dopamine and dobutamine.

Colforsin daropate is a recently developed water-soluble derivative of forskolin that directly stimulates adenylate cyclase, unlike the catecholamines. The chronotropic, inotropic and coronary vasodilator actions of colforsin daropate were compared with those of isoproterenol, dopamine and dobutamine, using canine isolated, blood-perfused heart preparations. The stimulating effect of each drug on adenylate cyclase activity was also assessed. Colforsin daropate, as well as each of the catecholamines, exerted positive chronotropic, inotropic and coronary vasodilator actions. The order of selectivity for the cardiovascular variables of colforsin daropate was coronary vasodilation > positive inotropy > positive chronotropy; whereas that of isoproterenol, dopamine and dobutamine was positive inotropy > coronary vasodilation > positive chronotropy. Thus, a marked characteristic of colforsin daropate is its potent coronary vasodilator action. On the other hand, each drug significantly increased the adenylate cyclase activity in a dose-related manner: colforsin daropate > isoproterenol > dopamine = dobutamine. These results suggest that colforsin daropate may be preferable in the treatment of severe heart failure where the coronary blood flow is reduced and beta-adrenoceptor-dependent signal transduction pathway is down-regulated.     

Adenylyl cyclase of perifused porcine granulosa cells remains responsive to pulsatile, but not continuous stimulation with follicle-stimulating hormone

Differentiation of granulosa cells and development of ovarian follicles requires FSH for several days. The purpose of the present studies was to determine how the capacity of the adenylyl cyclase of immature porcine granulosa cells to respond to FSH depends on the dose, duration, and frequency of exposure to FSH. Cells were stimulated with various regimens of FSH and forskolin in a dynamic flow perifusion system. cAMP production during 4 h of continuous (tonic) exposure to FSH was directly related to FSH concentration (5-500 ng/ml). FSH-stimulated cAMP production declined markedly after 4 h of tonic stimulation with FSH, regardless of the FSH concentration. Preliminary experiments using a pulse interval of 3 h (perifusion medium FSH concentration, 150 ng/ml) indicated that stimulation with 15-min pulses elicited a greater cumulative cAMP response than stimulation with either 5-, 30-, or 60-min pulses. Responsiveness to FSH depended critically on the duration and frequency of stimulation and the concentration of FSH. Short pulses were more effective than long pulses in both eliciting cAMP responses of most uniform amplitude and maintaining responsiveness to a final tonic FSH stimulus. The optimal pattern of stimulation consisted of a pulse duration of 15 min, with a pulse interval of 2-3 h. A peak chamber FSH concentration of 150 ng/ml yielded the greatest cumulative cAMP production, although cells that had been perifused with FSH-free medium had the greatest response to a final tonic FSH stimulus. The attenuation of responsiveness after continuous perifusion with FSH does not appear to be due to desensitization of the cyclase itself, since 1) cells perifused with FSH continuously for 20 h still responded to forskolin (100 microM), which activates cyclase independently of the FSH receptor; and 2) cells did not become refractory to forskolin for 14 h. The transient refractoriness to FSH appears to be due to a process that alters the interaction between the FSH receptor and the guanine nucleotide regulatory component of cyclase. This refractoriness can be reversed simply by removing FSH from the perifusion medium for a critical period of time, i.e. 2-3 h.     

On the mechanism of inhibition by iodine of the thyroid adenylate cyclase response to thyrotropic hormone.

Rats maintained on a low-iodine diet were hypophysectomized, and their diet was than enriched with iodide. Cyclic AMP (cAMP) concentrations achieved in their thyroids following in vitro TSH stimulation were significantly lower than those in the thyroids of control animals that did not receive dietary iodide enrichment. The addition of 0.1% methimazole (MMI) or 1% KC1O4 to the diet abolished this inhibitory effect of iodide. The administration of triiodothyronine in the died did not reproduce the inhibitory effect of iodide. The effect of iodide in vitro on the thyroid cAMP response to TSH was then investigated using paired thyroid lobes obtained from intact rats fed a low-iodine diet. During a 15-min incubation period, concentrations of iodide up to 10(-3)M, together with TSH (125 mU/ml), did not affect the thyroid cAMP response to TSH. In contrast, the preincubation of the lobes in 5 X 10(-5)M Nal for 2 h preceding a final 15-min incubation in medium containing TSH alone resulted in final cAMP concentrations significantly lower than those in paired lobes not exposed to iodide. Basal cAMP concentrations in thyroids not subjected to TSH stimulation were unaffected by preincubation in iodide. The inclusion of TSH during the preincubation period augmented the inhibitory effect of iodide on the final thyroid cAMP concentration achieved. The inclusion of MMI together with iodide during the preincubation period abolished the inhibitory effect of iodide on the final cAMP concentration achieved by TSH stimulation. Direct measurement of newly formed organic iodine in vitro demonstrated it to be inversely proportional to the final cAMP concentration achieved by TSH stimulation. The preincubation of thyroid lobes in iodide was without effect on the subsequent stimulation of cAMP by PGE1, or on the stimulation by F- of adenylate cyclase activity in the thyroid homogenate. The data support the concept of an as yet unknown organic form of iodine that limits thyroid adenylate cyclase responsiveness to TSH stimulation. This may, in part, explain the diverse, and generally inhibitory, actions of iodide on thyroid function.