Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Proctolin's role in neurally evoked contractions of the locust oviducts

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10⁻⁹ M and maxima at 2.5 × 10⁻⁸ M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 139–150, 1997     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-³H₂]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 ± 0.008 min ¹ using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 ± 0.00013 min ¹. An association rate constant of 7.3 × 10⁶ M ¹ min ¹ was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B_max = 23.87 ± 1.15 pmol/mg protein, K_d = 18.1 ± 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC₃₀ in parentheses): azadirachtin (1.55 × 10⁻⁸ M) > dihydroazadirachtin (3.16 × 10⁻⁸ M) > dansyl dihydroazadirachtin (7.40 × 10⁻⁸ M) > DNP-azadirachtin (7.50 × 10⁻⁸ M) > biotin dihydroazadirachtin (1.27 × 10⁻⁷ M) ≫ 11-methoxy 22,23-dihydroazadirachtin (6.67 × 10⁻⁷ M). Arch. Insect Biochem. Physiol. 34:461–473, 1997. © 1997 Wiley-Liss, Inc.     

Mode of action of proctolin on locust visceral muscle

Proctolin increases the frequency and amplitude of myogenic contractions and results in a sustained contraction of the oviducts of Locusta migratoria. The possible mode of action of proctolin receptors on this visceral muscle has been investigated. Calcium-free saline, containing either 20 mM magnesium ions or 100 μM EGTA, inhibited myogenic contractions, lowered basal tension, and abolished all the effects of proctolin following a 20 min incubation. These effects were reversible upon washing with normal saline. Similar results were obtained with normal saline containing 10 mM cobalt ions. Nifedipine at 50 μM lowered basal tension, abolished myogenic contractions, and reduced the proctolin-induced sustained contraction by 42-62% at 0.5 nM proctolin and by 33-37% at 5 nM proctolin. Similar results were obtained with 100 μM verapamil. Proctolin was still capable of eliciting considerable contractions (25-67% of controls) in preparations depolarized with 100 mM potassium saline. The removal of calcium from the high-potassium saline reversibly abolished the potassium-induced contraction and reversibly blocked the action of proctolin. Nifedipine was ineffective in blocking the action of proctolin in high-potassium saline. Neither cyclic AMP levels nor cyclic GMP levels of the lateral oviducts were elevated by proctolin in the presence of a phosphodiesterase inhibitor. The results indicate that proctolin mediates its effects via an influx of external calcium ions. This calcium appears to enter through two channels, a voltage-dependent channel and a receptor-operated channel. Cyclic nucleotides do not appear to be involved in the action of proctolin in this visceral muscle.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.     

Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor

1. Acetylcholine (ACh; 10⁻⁶ M—7 × 10⁻⁵ M), in the presence of neostigmine (10⁻⁵ M), caused contraction of the locust isolated foregut.2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine.3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10⁻⁶M) and reduced by d-tubocurarine (10⁻⁵ M) and decamethonium (5 × 10⁻⁵ M). Hexamethonium and α-bungaro-toxin had no effect on contractions caused by the above agonists.4. None of the antagonists used in this study blocked the contractile effects of nicotine.5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Effects of VA-045, a novel apovincaminic acid derivative,on isolated blood vessels: Cerebroarterial selectivity

We investigated the effects of VA-045, an apovincaminic acid derivative, on isolated blood vessels. VA-045 (10⁻⁷−10⁻⁵M) and vinpocetine (10⁻⁷−10⁻⁵M) inhibited the 64 mM KCl-induced and 10⁻⁶M norepinephrine (NE)-induced contraction of rat aortic strips. VA-045 (10⁻⁷−10⁻⁴M) and vinpocetine (10⁻⁷−10⁻⁴M) inhibited the activity of cyclic AMP and cyclic GMP phosphodiesterase in porcine coronary artery. VA-045 (3×10⁻⁹−3×10⁻⁶M) relaxed the 64 mM KCl-induced contraction of the canine basilar artery without affecting the peripheral arteries. These results indicate that VA-045 selectively dilates canine cerebral artery, and that it may be a useful agent for the treatment of cerebrovascular diseases such as stroke.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Acetylcholine-induced contractions in a holothurian (Isostichopus badionotus) smooth muscle are blocked by the calcium antagonists,diltiazem and verapamil

1. The longitudinal muscle of the body wall (LMBW) of the holothurian, Isostichopus badionotus contracted when treated with acetylcholine (ACh). The threshold concentration for initiating a contraction was 10⁻⁸M ACh.2. Inward calcium (Ca²⁺) current blockers, diltiazem and verapamil, blocked contractions induced by ACh suggesting that Ca²⁺ channels are involved. Verapamil caused small rhythmic contractions to occur in some muscle preparations.3. Caffeine initiated contractions only at the high concentration of 10 mM and caused rhythmic contractions in otherwise non-spontaneously beating muscle. The caffeine-contractions were partially blocked by verapamil.     

A novel response to propranolol: Contractile response in the isolated rabbit ear artery

Propranolol caused a contractile response in the isolated rabbit ear artery (EA). The concentration of propranolol causing a threshold contraction was 1.76 × 10^?6M while that causing a maximal contraction of 2.2 ± 0.18 g was 3 × 10^?5M. Higher concentrations caused tissue relaxation. Phentolamine, 10^?7 and 10^?6M reduced the propranolol-induced contractions by 50% and 90%, respectively while prazosin, 10^?8, 10^?7 and 10^?6M caused reductions of 54, 74 and 88%, respectively. Reserpinization of the rabbit with 5 mg/kg 24 hours before use eliminated the EA contractile response to tyramine but had no effect on that to propranolol. Desmethylimipramine plus deoxycorticosterone acetate enhanced the submaximal contraction of the EA to propranolol. $\text{In vitro}$ denervation with 6-hydroxydopamine (6-OHDA) decreased the response of the EA to tyramine and propranolol by 96% and 85% respectively but increased that to norepinephrine (NE) by 11%. Rabbit thoracic aorta (TA) did not respond to propranolol. In EA contracted with vasopressin o or 30 mM potassium, propranolol 10^?4 and 3 × 10^?4M caused a 20% and 100% relaxation, respectively. It is concluded that propranolol elicits a contractile response in the EA, at least in part, by direct activation of postsynaptic alpha adrenoceptors.     

Effect of angiotensin II receptor blocker on angiotensin II stimulated dna synthesis of cultured human aortic smooth muscle cells

To examine the role of the renin-angiotensin system on human vascular smooth muscle cell (VSMC) replication, we studied the effect of DUP753, an angiotensin II (ANG II) type 1 receptor antagonist, on ANG II stimulated tritiated-thymidine (³H-Tdr) incorporation into cultured human aortic VSMC. ANG II stimulated DNA synthesis of VSMC in a dose-dependent manner as estimated by ³H-Tdr incorporation (control; 2993 ± 486 cpm, 10⁻⁸M; 3360 ± 350 cpm, 10⁻⁷M; 3474 ± 516 cpm, 10⁻⁶M; 4889 ± 320 cpm, P < 0. 01). The effects of ANG II were clearly inhibited by 10⁻⁶M DUP 753 (ANG II 10⁻⁸M; 3360 ± 350 vs 509 ± 39 cpm, 10⁻⁷M; 3474 ± 516 vs 661 ± 36 cpm, 10⁻⁶M; 4889 ± 320 vs 806 ± 76 cpm, each P < 0. 01). This receptor antagonist decreased the basal ³H-Tdr incorporation of VSMC from 2933 ± 486 to 411 ±78 cpm (P < 0. 01). Furthermore, DUP 753 decreased 10⁻⁷M ANG II-stimulated ³H-Tdr incorporation of VSMC in a dose-dependent manner (control; 2627 ± 256 cpm, 10⁻⁹M; 2145 ± 143 cpm, 10⁻⁸M; 1047 ± 543 cpm, 10⁻⁷M; 639 ± 169 cpm, 10⁻⁶M; 642 ± 59 cpm, P < 0. 01). These observations suggest that, in human VSMC, ANG II type 1 receptors are important for the regulation of both stimulated and basal cell proliferation. It may therefore be worth while to examine the clinical usefulness of DUP 753 for preventing abnormal VSMC growth.     

Atrial natriuretic factor receptors and stimulation of cyclic GMP formation in normal and malignant osteoblasts

Synthetic rat atrial natriuretic factor (Ile-ANF-26) stimulated cyclic GMP formation by up to several hundred-fold in osteoblast-rich cultures from newborn rat calvaria and in clonal osteogenic sarcoma cells (UMR 106-01) which are phenotypically osteoblast. ANF had no effect on the cyclic AMP response to parathyroid hormone in the same cells. Specific, high-affinity binding sites for ANF were identified in both cell types, with K_d and receptor numbers in normal osteoblasts of 1.2 ± 0.1 × 10⁻¹⁰ M and 42 ± 4 × 10³ per cell, and in UMR 106-01 cells of 1.4 ± 0.1 × 10⁻¹⁰M and 22 ± 4 × 10³per cell.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

The oviduct musculature of the cockroach Leucophaea maderae and its response to various neurotransmitters and hormones

The musculature of the oviduct consists of an outer, irregular layer of longitudinal muscle and an inner layer of circular muscle. The four basic modes of activity—compression, segmentation, peristalsis, and reverse peristalsis—were evident in the isolated oviduct. These spontaneous events often occurred in an organized sequence. In fact eggs could be transported down the lateral oviducts by this myogenic activity once the sphincter between the common oviduct and vagina was severed. Myographic recordings were made of only the contractions of the longitudinal muscles. L-glutamate caused a distinct phasic contraction at 2.2 × 10^?5 M. The response became larger and more complex as the concentration of the amino acid was increased. Acetylcholine (1.6 × 10^?5 M) caused either a phasic or tonic response, or a combination thereof. By contrast, 5HT and tyramine simply increased the frequency of small phasic contractions, although in some preparations both monoamines caused an inhibition. The ecdysones, a juvenile hormone analogue (1 × 10^?6 M), and prostaglandin E₂ had no effect on oviduct activity. Initially high KCI solutions (162 mM) without Ca⁺⁺ induced a strong contraction but subsequent additions failed to do so. However, when a high KCI solution (158 mM) with 2 mM Ca⁺⁺ was added to the preparation the response was partially restored. Also the potent calcium antagonist Mn⁺⁺ (2mM) can suppress spontaneous activity.