Interaction between octopamine and proctolin on the oviducts of Locusta migratoria

The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3',5'-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10(-9) M proctolin-induced contraction was inhibited by 83% in the presence of 10(-5) M octopamine, and was completely inhibited in the presence of 10(-5) M octopamine plus 5x10(-4) M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10(-9) M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5x10(-6) M octopamine. These results indicate that octopamine and proctolin can antagonize each other's physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels.     

Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor

Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

Tyramine as a possible neurotransmitter/neuromodulator at the spermatheca of the African migratory locust, Locusta migratoria

Tyramine-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons projecting to the spermatheca of adult females of Locusta migratoria. Tyramine-like immunoreactive processes were also found throughout all regions of the spermatheca and tyramine-like immunoreactive bipolar or multipolar neurons were present on the spermathecal sac. HPLC coupled with electrochemical detection revealed more tyramine than octopamine present in spermathecal tissue. Electrical stimulation of the ventral ovipositor nerve resulted in a significant increase in calcium-dependent release of tyramine from the spermatheca. Both tyramine and octopamine increase the frequency and basal tonus of spermathecal contractions in a dose-dependent manner, with octopamine having a lower threshold. When tyramine is applied along with a half maximal octopamine dose, there is an additive effect on contractions of the spermatheca with slight synergistic effects at lower doses of tyramine. High concentrations of tyramine (10(-4)M) stimulated increases in cyclic AMP levels of the spermatheca; an effect blocked by phentolamine. Phentolamine has a higher affinity (and thus a lower IC(50) value congruent with5.6x10(-8)M) than yohimbine (IC(50) congruent with1.1x10(-4)M) in reducing tyramine-induced spermathecal contractions. Taken together, these results suggest that tyramine may be a co-transmitter with octopamine at the spermatheca, with both neuroactive chemicals acting on an octopamine receptor.     

Effects of maturation on synaptic potentials in the locust flight system

We have measured parameters of identified excitatory postsynaptic potentials from flight interneurons in immature and mature adult locusts (Locusta migratoria) to determine whether parameters change during imaginal maturation. The presynaptic cell was the forewing stretch receptor. The postsynaptic cells were flight interneurons that were filled with Lucifer Yellow and identified by their morphology. Excitatory postsynaptic potentials from different postsynaptic cells had characteristic amplitudes. The amplitude, time to peak, duration at half amplitude and the area above the baseline of excitatory postsynaptic potentials did not change with maturation. The latency from action potentials in the forewing stretch receptor to onset of excitatory postsynaptic potentials decreased significantly with maturation. We suggest this was due to an increase in conduction velocity of the forewing stretch receptor. We also measured morphological parameters of the postsynaptic cells and found that they increased in size with maturation. Growth of the postsynaptic cell should cause excitatory postsynaptic potential amplitude to decrease as a result of a decrease in input resistance, however, this was not the case. Excitatory postsynaptic potentials in immature locusts depress more than in mature locusts at high frequencies of presynaptic action potentials. This difference in frequency sensitivity of the immature excitatory postsynaptic potentials may account in part for maturation of the locust flight rhythm generator.Abbreviations EPSP excitatory postsynaptic potential - fSR forewing stretch receptor - IPSP inhibitory postsynaptic potential - SR stretch receptor     

Spontaneous activity of foregut and hindgut visceral muscle of the locust,Locusta migratoria—II. The effect of biogenic amines

1. At 10⁻⁸ M, 5-HT increased both the frequency and amplitude of contractions of isolated locust foregut. At 10⁻⁴ M the foregut general tonus was increased.2. Both spontaneously active and quiescent hindguts were less sensitive to 5-HT, showing only an increase in amplitude of contraction at 10⁻⁶−10⁻⁵ M.3. The Hill plot suggested that although the 5-HT receptor populations in these two gut divisions differed in affinities, they were essentially homogeneous.4. Octopamine (10⁻⁵−10⁻⁴ M) increased foregut contraction frequency but diminished amplitude.5. Octopamine action on the hindgut was varied. At 10⁻⁸ M it slightly increased tonus, while at 10⁻⁵ M it increased contraction amplitude without affecting frequency.6. At 10⁻⁴ M octopamine suppressed activity of spontaneously active preparations and lowered the tonus of quiescent preparations.7. Tyramine induced dose-dependent inhibition of foregut responses to 5-HT. The hindgut was exceptionally sensitive to tyramine, at only 10⁻⁸ M it suppressed 5-HT responses.8. Octopamine inhibited fore- and hindgut responses to 5-HT, but was less effective than tyramine.9. Locust fore- and hindgut have remarkably different pharmacological properties reflecting differences in innervation and in extrajunctional monoamine receptor affinities.     

A comparison of the signalling properties of two tyramine receptors from Drosophila

In invertebrates, the phenolamines, tyramine and octopamine, mediate many functional roles usually associated with the catecholamines, noradrenaline and adrenaline, in vertebrates. The α‐ and β‐adrenergic classes of insect octopamine receptor are better activated by octopamine than tyramine. Similarly, the Tyramine 1 subgroup of receptors (or Octopamine/Tyramine receptors) are better activated by tyramine than octopamine. However, recently, a new Tyramine 2 subgroup of receptors was identified, which appears to be activated highly preferentially by tyramine. We examined immunocytochemically the ability of CG7431, the founding member of this subgroup from Drosophila melanogaster, to be internalized in transfected Chinese hamster ovary (CHO) cells by different agonists. It was only internalized after activation by tyramine. Conversely, the structurally related receptor, CG16766, was internalized by a number of biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline, which also were able to elevate cyclic AMP levels. Studies with synthetic agonists and antagonists confirm that CG16766 has a different pharmacological profile to that of CG7431. Species orthologues of CG16766 were only found in Drosophila species, whereas orthologues of CG7431 could be identified in the genomes of a number of insect species. We propose that CG16766 represents a new group of tyramine receptors, which we have designated the Tyramine 3 receptors.     

Effect of forskolin and VIP on the release of somatostatin and the cAMP content of cultured rat diencephalon neurons

Forskolin, a direct activator of adenylate cyclase, stimulates somatostatin release in dispersed fetal diencephalic cells in culture (10 j). It was found that concentrations ranging from 10(-8) M to 10(-4) M increase the release of somatostatin in a dose-dependent manner, as well as the formation of intracellular cyclic AMP. Furthermore, VIP (10(-6) M) which produces a significant (p less than 0.03) elevation of SRIF release at 30 min of incubation, also induces a prompt increase of intracellular cyclic AMP (10 min). These results suggest that VIP could stimulate the somatostatin release through a cyclic AMP-dependent mechanism.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

Crustacean cardioactive peptide is a modulator of oviduct contractions in Locusta migratoria

Crustacean cardioactive peptide (CCAP) stimulates the contractions of locust oviducts. CCAP increased the basal tonus and increased the frequency and amplitude of phasic contractions, as well as the amplitude of neurally-evoked oviduct contractions in a dose-dependent manner. Oviducts from Vth instar larvae and adult locusts aged 10 days or less, were more sensitive to CCAP than oviducts from adult locusts aged 12 days or more. This may be indicative of a differential expression of number or subtypes of CCAP receptors on the oviducts at different ages, and may be related to reproductive functions or to functions of CCAP on the oviducts during ecdysis. The oviducts appear more sensitive to CCAP when compared with previously published reports of CCAP actions on the hindgut. CCAP actions on the amplitude of neurally-evoked contractions of the oviducts are similar to those of proctolin, however, the oviducts are more sensitive to CCAP. No CCAP-like immunoreactive structures were discovered in the nerves innervating the oviducts, or on the oviducts themselves, confirming the previously published suggestion (Dircksen et al., 1991) that CCAP acts as a neurohormone at the oviducts. Cells showing CCAP-like immunoreactivity were discovered in the fat body associated with the oviducts and represent a potential source of CCAP, along with CCAP released from the transverse nerve and perivisceral organs.     

A neurohormonal role for serotonin in the control of locust oviducts

Serotonin increases the frequency and amplitude of spontaneous contractions and leads to an increase in the basal tonus of the locust oviducts. These effects were dose-dependent and were seen on both the non-innnervated and innervated portion of the oviducts. Vertebrate type serotonin agonists and antagonists were used and the profile shows that the receptors on the non-innervated and innervated portion of the oviducts are more similar to 5-HT3 receptors than to either 5-HT1 or 5-HT2 receptors. No serotonin was found associated with the oviducts or the innervation to the oviducts using immunohistochemistry and HPLC coupled to electrochemical detection, suggesting a neurohormonal role for serotonin in the control of locust oviducts.     

Characterization of the tyraminergic system in the central nervous system of the locust,Locusta migratoria migratoides

Tyramine occurs in the central nervous system (CNS) of the migratory locust,Locusta migratoria migratoides. The distribution of tyramine within the CNS does not parallel that of octopamine. Tyramine is synthesised from tyrosine in the presence of tyrosine decarboxylase. A second decarboxylase in the CNS is active against 5HTP and DOPA. The locust ganglia incorporate tyramine by high- and low-affinity uptake processes that appear to be independent of dopamine and octopamine. Depolarisation of the locust ganglia by high potassium concentration results in calcium-dependent release of incorporated [³H]tyramine.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

Signal transduction for Schistocerca gregaria ion transport peptide is mediated via both cyclic AMP and cyclic GMP

The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl⁻, K⁺ and Na⁺ reabsorption, whereas secretion of H⁺ into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl⁻ uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

Calcium rather than cyclic AMP is an intracellular messenger of parathyroid hormone action on glycogen metabolism in isolated rat hepatocytes.

The synthetic 1-34 fragment of human parathyroid hormone (1-34hPTH) stimulated glucose production in isolated rat hepatocytes. The effect of 1-34hPTH was dose-dependent and 10(10) M-1-34 hPTH elicited the maximum glucose output, which was approx. 80% of that by glucagon. Although 1-34hPTH induced a small increase in cyclic AMP production at concentrations higher than 10(-9) M, 10(-10) M-1-34hPTH induced the maximum glucose output without significant elevation of cyclic AMP. This is in contrast to the action of forskolin, which increased glucose output to the same extent as 10(-10) M-1-34hPTH by causing a 2-fold elevation of cyclic AMP. In addition to increasing cyclic AMP, 1-34hPTH caused an increase in cytoplasmic free calcium concentration ([Ca2+]c). When the effect of 1-34hPTH on [Ca2+]c was studied in aequorin-loaded cells, low concentrations of 1-34hPTH increased [Ca2+]c: the 1-34hPTH effect on [Ca2+]c was detected at as low as 10(-12) M and increased in a dose-dependent manner. 1-34hPTH increased [Ca2+]c even in the presence of 1 microM extracellular calcium, suggesting that PTH mobilizes calcium from an intracellular pool. In line with these observations, 1-34hPTH increased the production of inositol trisphosphate. These results suggest that: (1) PTH activates both cyclic AMP and calcium messenger systems and (2) PTH stimulates glycogenolysis mainly via the calcium messenger system.     

Glutamate potential : differences from the excitatory junctional potential revealed by diltiazem and concanavalin A in crayfish neuromuscular junction.

1. The effect of diltiazem and concanavalin A (Con A) on the crayfish neuromuscular junction was investigated in order to compare the action of L-glutamate with that of the excitatory transmitter. 2. When diltiazem (0.3 nM) was added to the perfusion fluid, the iontophoretic glutamate potential was reduced to about half, whereas the amplitude of excitatory junctional potentials (EJPs) increased by about two times. 3. Dose-response curves of L-glutamate suggested that diltiazem acted in a non-competitive manner. The decrease in amplitude of the glutamate potential caused by diltiazem was not due to the acceleration of desensitization of the glutamate receptor. 4. The increase in amplitude of EJPs caused by diltiazem was due to the increase in membrane resistance. The quantal content and size of extracellular EJPs were not affected by diltiazem. 5. In normal saline, bath application of glutamate decreased the amplitude of both glutamate potentials and EJPs because of desensitization of the glutamate receptor. The decrease in amplitude of the glutamate potential was completely prevented by previous application of Con A (10(-6) M). On the other hand, Con A had no influence on the decrease in amplitude of EJPs. 6. Some possible explanations of these pharmacological differences between glutamate potentials and EJPs revealed by diltiazem and Con A are considered.     

Mechanisms of the antilipolytic response of human adipocytes to tyramine,a trace amine present in food

Tyramine is found in foodstuffs, the richest being cheeses, sausages, and wines. Tyramine has been recognized to release catecholamines from nerve endings and to trigger hypertensive reaction. Thereby, tyramine-free diet is recommended for depressed patients treated with irreversible inhibitors of monoamine oxidases (MAO) to limit the risk of hypertension. Tyramine is a substrate of amine oxidases and also an agonist at trace amine-associated receptors. Our aim was to characterize the dose-dependent effects of tyramine on human adipocyte metabolic functions. Lipolytic activity was determined in adipocytes from human subcutaneous abdominal adipose tissue. Glycerol release was increased by a fourfold factor with classical lipolytic agents (1 μM isoprenaline, 1 mM isobutylmethylxanthine) while the amine was ineffective from 0.01 to 100 μM and hardly stimulatory at 1 mM. Tyramine exhibited a partial antilipolytic effect at 100 μM and 1 mM, which was similar to that of insulin but weaker than that obtained with agonists at purinergic A1 receptors, α₂-adrenoceptors, or nicotinic acid receptors. Gi-protein blockade by Pertussis toxin abolished all these antilipolytic responses save that of tyramine. Indeed, tyramine antilipolytic effect was impaired by MAO-A inhibition. Tyramine inhibited protein tyrosine phosphatase activities in a manner sensitive to ascorbic acid and amine oxidase inhibitors. Thus, millimolar tyramine restrained lipolysis via the hydrogen peroxide it generates when oxidized by MAO. Since tyramine plasma levels have been reported to reach 0.2 μM after ingestion of 200 mg tyramine in healthy individuals, the direct effects we observed in vitro on adipocytes could be nutritionally relevant only when the MAO-dependent hepato-intestinal detoxifying system is overpassed.     

HS-142-1, a Novel Non-peptide Antagonist for Atrial Natriuretic Peptide Receptor,Selectively Inhibits Particulate Guanylyl Cyclase and Lowers Cyclic GMP in LLC-PK1 Cells

HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK₁ cells. At concentrations from 0.1 to 100 μg/ml (= 2.5 × 10^–8 – 2.5 × 10^–5 M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10^–8 M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10^–5 M sodium nitroprusside. HS–142–1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulation of cyclic GMP in LLC-PK₁ cells through interacting with guanylyl cyclase-linked receptors.     

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.