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PubMed1984

Inhibition of the thromboplastin response of endothelial cells in vitro.

Confluent monolayers of human umbilical vein endothelial cells in culture responded with a 5-fold increase in thromboplastin (tissue factor) synthesis when exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (50 ng/ml) or endotoxin (ETX) (25 micrograms/ml) for 16 hr. This induced thromboplastin synthesis was markedly inhibited by exposure of the cells to two different phosphodiesterase inhibitors, methylisobutylxanthine (MIX) and rac-4(3-butoxy-4-methoxybenzyl)-2-imidazole idinone (RO-20-1724) and to the transmethylation inhibitors 3-deazaadenosine (DZA) and 1-homocysteine thiolactone (Hcy) in combination. It was slightly (TPA) or not at all (ETX) inhibited upon exposure of the cells to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4, 5-trimethoxybenzoate hydrochloride (TMB-8). However, in the presence of MIX TMB-8 had a moderate additional inhibitory effect on TPA-induced thromboplastin response. The thromboplastin response of endothelial cells in vitro thus probably depends on transmethylation events for its full expression. It is also strongly modulated by the intracellular level of cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Reactivation of carboxylester hydrolase following inhibition by 4-nitrophenyl organophosphinates.

Porcine liver carboxylester hydrolase (EC 3.1.1.1; carboxylesterase) was rapidly inhibited by 4-nitrophenyl organophosphinates containing aryl or heteroaryl groups directly bound to phosphorus. The most potent inhibitor was 4-nitrophenyl di-2-thienylphosphinate for which the median inhibitory concentration was 7.4 X 10(-9) M. Rabbit liver monomeric carboxylester hydrolase was inhibited, separated from excess inhibitor by gel permeation chromatography, and observed for spontaneous or 1,1'-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-4)-induced reactivation. Recovery was most rapid (k = 4 to 7 X 10(-4) min-1) from phosphinyl groups containing one alkyl substituent smaller than isopropyl and one aryl or heteroaryl group smaller than naphthyl. The di-2-thienylphosphinylated enzyme was an exception since it recovered rapidly while lacking an alkyl substituent. Oxime reactivation by TMB-4 doubled rates of recovery.

Animals↗
PubMed1986

Effect of some drugs on thromboplastin activity in mouse trophoblast cells in vitro and in vivo.

Mouse trophoblast cells are constitutive producers of the thromboplastin apoprotein in vitro. The effects on thromboplastin activity of the three transmethylation inhibitors 3-deazaadenosine (DZA), 3-deazaaristeromycin (DZAri) and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), the four calcium antagonists TMB-8, verapamil, nifedipine and felodipine, the prostaglandin E2 (PGE2), the phosphodiesterase inhibitor 1-methyl 3-isobutylxanthine (MIX) and monensin have been studied. No cytotoxic effects were detected when trypan blue exclusion, release of lactic dehydrogenase, incorporation of 14C-leucine into protein and cell morphology were monitored. TMB-8, felodipine, nifedipine and verapamil all abolished the increase in thromboplastin when added after 68 hr or 90-96 hr in culture. EHNA and DZAri had the same effect (but were only added at 90-96 hr). DZA had a similar effect when added at 68 hr and an even more marked inhibitory effect when added at 90-96 hr. Monensin prevented the increase in thromboplastin activity at 68 hr as well as at 90-96 hr. The combination of DZA and 1-homocysteine thiolactone (Hcy) further increased the inhibition, indicating that in these cases synthesis as well as degradation of thromboplastin were altered. The combination of DZA/Hcy and one of the four calcium antagonists gave no additional inhibitory effect. PGE2 had a biphasic dose-dependent effect. The increased thromboplastin activity at low concentrations of PGE2 (10 ng/ml) was inhibited by addition of one of the compounds verapamil, felodipine, nifedipine or DZA/Hcy. PGE2 at higher levels (10 micrograms/ml) significantly inhibited thromboplastin synthesis. Combination of PGE2 (10 micrograms/ml) and one of the calcium antagonists, DZA/Hcy or MIX gave no significant additive inhibitory effect.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Thrombin-stimulated phosphorylation of myosin light chain and its possible involvement in endothelin-1 secretion from porcine aortic endothelial cells.

Thrombin-stimulated secretion of endothelin-1 (ET-1) from porcine aortic endothelial cells was inhibited in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) also prevented the thrombin-stimulated secretion of ET-1 but it enhanced the accumulation of ET-1 in the endothelial cells. When the endothelial cells were treated with thrombin, the phosphorylation of a 20-kDa protein which was identified as myosin light chain (MLC) was detected. Phosphorylation was augmented in a time-dependent manner. As in the case of ET-1 secretion, MLC phosphorylation was prevented by TMB-8, trifluoperazine, W-7 and ML-9 at the same concentrations which were effective in inhibiting the ET-1 secretion. The site of phosphorylation of MLC was identified as a serine residue. Parallel to the phosphorylation of MLC, thrombin increased the amounts of the 43- and 200-kDa proteins in the Triton-insoluble fraction; these proteins were identified as actin and myosin heavy chain, respectively. These results suggest that the MLC phosphorylation elicited by MLC kinase may facilitate the formation of filamentous myosin and actin which are probably involved in ET-1 secretion, possibly in the transport of ET-1-containing vesicles in thrombin-stimulated endothelial cells.

Animals↗
PubMed1980

Brain stem projections of the aortic nerve in the cat: a study using tetramethyl benzidine as the substrate for horseradish peroxidase.

The intra-axonal transport of horseradish peroxidase (HRP) has been used to trace the nodose ganglion and brain stem projections of a physiologically distinct nerve - the aortic depressor nerve - following electrophysiological identification. Tetramethyl benzidine (TMB) has been used as the substrate for demonstrating the centrally transported HRP15, 16. This sensitive method for horseradish peroxidase histochemistry has permitted the visualization of the central projections of aortic nerve afferents and has also provided information regarding the anatomical localization of cell bodies of these sensory nerve fibers within the nodose ganglion. This study demonstrates the usefulness of using TMB as a substrate for HRP histochemistry in anatomical studies where the detection of anterogradely transported HRP is an essential prerequisite. The uptake of HRP from the cut central ends of sensory nerve fibers and the transport of this enzyme to the sensory ganglion and subsequently into the central processes of these sensory neurons have made possible this study of the central projections of a functionally distinct peripheral nerve. Information has been provided by this study that cell bodies of aortic nerve afferent fibers are localized in the rostrolateral pole of the nodose ganglion. Dense central projections of sensory terminals of aortic afferents have been found in the dorsolateral and medial subdivisions of the nucleus of the tractus solitarius. These central projections of aortic afferents extend for 6 mm rostrocaudally in the medulla with the densest projection being found at the level of the obex. These projections are bilateral at all rostrocaudal levels. This anatomical demonstration of the dorsolateral and medial subdivisions of the nucleus of the tractus solitarius confirms earlier reports based on electrophysiological studies. Of particular interest in this study is the new observation that there exists a dense projection of aortic nerve afferents to the area postrema. The possible physiological implications of a direct input of peripheral chemoreceptor afferents to a region of central chemosensitivity are discussed. The complete absence of any retrogradely labeled cell body in the brain stem from exposure of the aortic nerve to horseradish peroxidase is noteworthy. This indicates that the aortic nerve is purely afferent in function and that reflex control of afferent activity in the aortic nerve is not mediated by brain stem neurons projecting down the same nerve.

Animals↗
PubMed1985

Evidence for a 'paravascular' fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space.

The protein tracer, horseradish peroxidase (HRP), was infused into the lateral cerebral ventricles or subarachnoid space of anesthetized cats and dogs after insertion of a cisternal cannula to permit drainage of cerebrospinal fluid (CSF) and tracer solution. The intracerebral distribution of the tracer was then determined by light microscopy of serial brain sections after postinfusion intervals of 4 min-2 h. For the localization of HRP, sections were incubated with diaminobenzidine (DAB) or the much more sensitive chromogen, tetramethylbenzidine (TMB). The TMB reaction showed a consistent 'paravascular' distribution of tracer reaction product, within the perivascular spaces (PVS) around large penetrating vessels and in the basal laminae around capillaries, far beyond the termination of the PVS. After infusion of HRP over 4 min, arterioles were surrounded by the tracer, but capillaries and venules were usually less densely demarcated; by 6 min, however, the intraparenchymal microvasculature was outlined in toto throughout the forebrain and brainstem. Electron microscopy of sections incubated in DAB after 10 or 20 min HRP circulation confirmed the paravascular location of the reaction product, which was also dispersed throughout the extracellular spaces (ECS) of the adjacent parenchyma. Our results demonstrate that solutes in the CSF have access to the ECS throughout the neuraxis within minutes via fluid pathways paralleling the intraparenchymal vasculature. The rapid paravascular influx of HRP could be prevented by stopping or diminishing the pulsations of the cerebral arteries by aortic occlusion or by partial ligation of the brachiocephalic artery. The exchange of solutes between the CSF and the cerebral ECS has generally been attributed to diffusion, however, HRP enters the neuraxis along the intraparenchymal microvasculature far more rapidly than can be explained on this basis. This apparent convective tracer influx may be facilitated by transmission of the pulsations of the cerebral arteries to the microvasculature. We postulate that a fluid circulation through the CNS occurs via paravascular pathways.

Anesthesia, General↗
PubMed1989

Evidence for a morphologically distinct subpopulation of striatipetal axons following injections of WGA-HRP into the ventral tegmental area in the rat.

Following injections of small volumes (10-30 nl) of WGA-HRP (1-2%) into the ventral tegmental area, axonal transport of the lectin-peroxidase conjugate to ventral striatum was evaluated by light microscopy after TMB histochemistry and by electron microscopy following stabilization of the TMB reaction product with DAB and H2O2. Label was distributed more or less evenly in ventral striatum, with only slight patchiness observable in the boundary zone between the nucleus accumbens and ventromedial caudate-putamen. The electron microscope revealed that labeled axons contained markedly flattened vesicles and dense axoplasm and contacted perikarya, dendrites and dendritic spines of short (0.2-0.3 microns) symmetric appositions. Boutons with a similar triad of morphological features were observed in preparations processed for conventional electron microscopy and for tyrosine hydroxylase immunocytochemistry, suggesting that the characteristic morphological features observed are not an epiphenomenon related to histochemical processing.

Animals↗
PubMed1992

Estrogen receptive neurons in the preoptic area of the rat are postsynaptic targets of a sexually dimorphic enkephalinergic fiber plexus.

The periventricular preoptic area (pePOA) is a sexually dimorphic component of the rat forebrain that contains a sexually dimorphic Met-enkephalin immunoreactive (ENK-ir) fiber plexus. This plexus is especially dense in the female while only scattered ENK-ir fibers are present in the pePOA of the male. Abundant estrogen receptive neurons are located in the pePOA of both the female and male. This experiment was conducted to determine if estrogen receptive neurons in the pePOA are postsynaptic targets of ENK-ir terminals. Double label ultrastructural localization of estrogen receptor (ER)-ir neurons and ENK-ir fibers was performed using the chromogens 3,3',5,5'-tetramethylbenzidine (TMB) and diaminobenzidine tetrahydrochloride (DAB), respectively. TMB-stained ER-ir neurons contained electron dense crystalline spicules located predominantly in their nuclei. Flocculent DAB reaction product was distributed over membraneous structures in ENK-ir fibers and terminals. Numerous ER-ir neurons were present in the pePOA of the male and female. In females, many ENK-ir terminals, both synaptic and non-synaptic, contacted the perikarya of ER-ir neurons. In contrast, many fewer ENK-ir terminals made contact on ER-ir neurons in the male. Thus, these results provide morphological evidence that ENK-ir neurons can regulate ER-ir neurons in the pePOA. Moreover, because expression of the ENK-ir pePOA fiber plexus is estrogen-sensitive in the female, these results suggest strongly that estrogen may regulate these neurons both pre- and postsynaptically. Finally, these results provide additional evidence for the involvement of the sexually dimorphic pePOA ENK-ir fibers plexus in the control of estrogen-mediated function in the female.

Animals↗
PubMed1995

Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat.

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support of this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

Animals↗
PubMed1986

Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing.

The concentration of cytosolic-free calcium (Ca2+i) was determined with aequorin in RAW-264 macrophage-like cells activated in vitro for tumor cell killing with lymphokine (LK) and lipopolysaccharide (LPS). Treatments of these cells with optimal doses of stimulants, which evoked the development of cytolytic activity, also induced a rise in their Ca2+i. No rise in Ca2+i could be observed under treatments which failed to activate cells. The presence of both stimulants was an absolute requirement for evoking cytolytic activity and also a rise in Ca2+i. There was an apparent parallelism between the rate of activation and the rate of rise in Ca2+i. Cells which slowly developed their cytolytic activity exhibited a slow rise in Ca2+i, while macrophages which acquired their cytolytic activity at the faster rate also showed a more rapid increase in Ca2+i. The development of cytolytic activity in RAW-264 macrophages was inhibited by two intracellular calcium antagonists, TMB-8 and ruthenium red. This inhibition could be reversed by high concentrations of extracellular calcium. TMB-8, at the concentrations which were effective in inhibiting the activation process, also completely blocked the associated rise in Ca2+i. These results suggested that Ca2+i might play a role in the mechanism of tumoricidal transformation of RAW-264 macrophages.

Aequorin↗
PubMed1987

Peroxidative metabolism of benzidine derivatives by Salmonella typhimurium.

Benzidine and several derivatives are activated to mutagenic species in an H2O2-dependent Ames test system. Optical and electron paramagnetic resonance (EPR) spectroscopy are employed in studies of the H2O2-dependent oxidation of benzidine and 3,5,3',5'-tetramethylbenzidine (TMB) catalyzed by intact bacteria, and provide direct evidence for peroxidase activity in Salmonella typhimurium. The acetylase-proficient Ames tester strain TA98 and its acetylase-deficient derivative TA98/1,8-DNP6 are equally responsive to H2O2-dependent mutagenicity; enzymatic acetylation appears not to be involved in activation of benzidine, in this system. The H2O2-dependent mutagenicity of benzidine and oxidation of TMB are observed when the assays are carried out in acetate buffer (pH 5.5), but not in 2-[N-morpholino]ethane sulfonic acid (MES) buffer, at the same pH. This difference is interpreted in terms of the effects of these buffers on the intracellular pH of the bacteria. The H2O2-dependent mutagenicity of several benzidine congeners is also described.

Benzidines↗
PubMed1985

In vitro inhibition of allergic histamine release by calcium antagonists.

The ability of calcium entry blockers to inhibit allergic histamine release from rabbit leukocytes was studied. Bepridil, verapamil, nifedipine, diltiazem and TMB-8 produced concentration-dependent inhibition of allergic histamine release from rabbit leukocytes. The calculated IC50s (microM) were as follows: verapamil = 1.3; bepridil = 2.3; TMB-8 = 3.0; nifedipine = 3.3; and diltiazem = 5.3. Verapamil also exerted concentration-dependent inhibition of allergic histamine release from human basophils with an IC50 of 3.7 microM. These agents may act by interfering with the influx of Ca2+ into the leukocytes as well as calcium-dependent steps (e.g., activation of calmodulin, phospholipase A2 and/or 5-lipoxygenase etc.) in the process of histamine secretion.

Animals↗
PubMed1992

Effect of Ca2+ modulators on acetylcholine-induced phasic and tonic contractions and A23187-induced contractions in ileal longitudinal muscle and IP3 production.

The influence of different sources of Ca2+ on the Emax and ED50 values of acetylcholine (ACh)-induced phasic and tonic contractions and on A23187-induced contractions was studied using different extracellular Ca2+ concentrations ([Ca2+]o) and the Ca2+ modulators TMB-8 and D600. IP3 production induced by both stimulants was also studied. The results are compatible with: (a) the mobilization of Ca2+ from an intracellular source as a primary event in the phasic response. (b) The primary involvement of a D600-sensitive inward Ca2+ current in the ACh-tonic response. (c) An inward D600-sensitive Ca2+ current associated with the ionophore transported ion. (d) The involvement of an IP3 independent, TMB-8 sensitive mechanism of Ca2+ mobilization involved in the A23187-induced responses.

Acetylcholine↗
PubMed1992

Pharmacological distinction of the hyperpolarization response to caffeine and acetylcholine in guinea-pig coronary endothelial cells.

Membrane potential changes in endothelial cells in response to caffeine and acetylcholine (ACh) were recorded with microelectrodes from an intact endothelium preparation from the guinea-pig coronary artery. Caffeine induced a transient hyperpolarization of the membrane in a concentration-dependent manner. The hyperpolarization was inhibited by removal of Ca2+ from the bathing medium and by ryanodine (20 microM). It was not affected by 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester hydrochloride (TMB-8, 10 microM) or neomycin (5 mM). ACh induced a sustained hyperpolarization in endothelial cells. At concentrations that caused no significant effects on the caffeine response, TMB-8 and neomycin inhibited hyperpolarization induced by ACh. Ryanodine did not inhibit the response to ACh. The ACh-induced hyperpolarization was also inhibited by caffeine in a concentration-dependent manner. Results from the present study suggest that hyperpolarizations induced by caffeine and ACh are mediated by separate Ca2+ pools.

Acetylcholine↗
PubMed1986

Role of calcium in luteinizing hormone-induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).

The effects of calcium on steroidogenesis and cyclic AMP production in chicken granulosa cells were examined. For the expression of full steroidogenic effects by LH, forskolin, and 8-bromo cyclic AMP the presence of calcium in the incubation medium was essential, the optimal concentration being 1 mM. Moreover, calcium antagonists (verapamil, TMB-8) significantly suppressed steroidogenesis in response to all three agonists. The metabolism of 25-hydroxycholesterol and the conversion of pregnenolone to progesterone, however, were not affected by the lack of calcium or by coincubation with calcium antagonists. LH-stimulated cyclic AMP production was also suppressed in calcium-deficient medium and in the presence of the putative calcium channel blocker, verapamil. However, TMB-8 did not affect LH-induced cyclic AMP production. Moreover, neither forskolin- nor IBMX-induced cyclic AMP accumulation was significantly affected by lack of calcium or verapamil. These results are interpreted to indicate that the continuous presence of extracellular calcium is essential for hormonal regulation of steroidogenesis and is important for events both proximal and distal to cyclic AMP generation up to pregnenolone synthesis.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Gamma irradiation as a substitute for H2O2 in peroxidase-labelled enzyme immunoassays.

The ability of gamma irradiation to substitute for H2O2 in peroxidase-labelled enzyme immunoassays was investigated. Colouring of TMB (3,3',5,5'-tetramethylbenzidine), ABTS [2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid)], and OPD (o-phenylenediamine) was produced with doses of 10 and 20 Gy. Addition of superoxide dismutase enhanced TMB and ABTS, and inhibited OPD. The reaction was terminated when the irradiation was stopped. The use of denaturing compounds to stop the reaction was unnecessary and coated antigens could be re-used for a second immunoassay.

Free Radicals↗
PubMed1991

A comparison of the sensitivity and specificity of enzyme immunoassays and time-resolved fluoroimmunoassay.

Time-resolved fluoroimmunoassay (TR-FIA) and various enzyme immunoassays (EIA) were compared in order to determine the detection system which showed the greatest degree of sensitivity without sacrificing specificity. The system chosen for the evaluation of these assays was the detection of antibodies to human immunodeficiency virus (HIV). For EIA, horseradish peroxidase (HRP) and alkaline phosphatase (AP) were investigated, each with a number of different substrates. HRP with its fluorogenic substrate, 3-(p-hydroxyphenyl)propionic acid (HPPA) was 1.6 times (p less than 0.01) more sensitive than with 3,3',5,5'-tetramethylbenzidine (TMB) and four times (p less than 0.001) more sensitive than with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). AP with its fluorogenic substrate, 4-methylumbelliferyl phosphate (4MeUP), was 6-7 times (p less than 0.001) more sensitive than with phenolphthalein monophosphate (PMP) and 8-13 times (p less than 0.001) more sensitive than with p-nitrophenyl phosphate (pNPP). TR-FIA with Eu3(+)-labelled anti-human IgG was equivalent in sensitivity to HRP with TMB and AP with 4MeUP.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Differential effects of calcium on progesterone production in small and large bovine luteal cells.

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗