Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Rhythmic activities of hypothalamic magnocellular neurons: Autocontrol mechanisms

Electrophysiological recordings in lactating rats show that oxytocin (OT) and vasopressin (AVP) neurons exhibit specific patterns of activities in relation to peripheral stimuli: periodic bursting firing for OT neurons during suckling, phasic firing for AVP neurons during hyperosmolarity (systemic injection of hypertonic saline). These activities are autocontrolled by OT and AVP released somato-dentritically within the hypothalamic magnocellular nuclei. In vivo, OT enhances the amplitude and frequency of bursts, an effect accompanied with an increase in basal firing rate. However, the characteristics of firing change as facilitation proceeds: the spike patterns become very irregular with clusters of spikes spaced by long silences; the firing rate is highly variable and clearly oscillates before facilitated bursts. This unstable behaviour dramatically decreases during intense tonic activation which temporarily interrupts bursting, and could therefore be a prerequisite for bursting. In vivo, the effects of AVP depend on the initial firing pattern of AVP neurons: AVP excites weakly active neurons (increasing duration of active periods and decreasing silences), inhibits highly active neurons, and does not affect neurons with intermediate phasic activity. AVP brings the entire population of AVP neurons to discharge with a medium phasic activity characterised by periods of firing and silence lasting 20–40 s, a pattern shown to optimise the release of AVP from the neurohypophysis. Each of the peptides (OT or AVP) induces an increase in intracellular Ca²⁺ concentration, specifically in the neurons containing either OT or AVP respectively. OT evokes the release of Ca²⁺ from IP3-sensitive intracellular stores. AVP induces an influx of Ca²⁺ through voltage-dependent Ca²⁺ channels of T-, L- and N-types. We postulate that the facilitatory autocontrol of OT and AVP neurons could be mediated by Ca²⁺ known to play a key role in the control of the patterns of phasic neurons.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Blockade by nickel ions of phasic contraction to K+ and high affinity calcium of ileal longitudinal muscle of guinea-pig

1. Preincubation with 1 or 2mM Ni²⁺ inhibited dose-dependently the ileal phasic response to K⁺ (60 mM) without appreciable effects on the tonic response. Ni²⁺ above 3mM inhibited the tonic response.2. Ni²⁺ inhibited the high affinity Ca²⁺ sites than the low affinity sites during K⁺ contraction.3. After treatment with Ni²⁺, the K⁺ response was fairly restored by a wash with normal medium. The nickel bound to the ileal cells was almost eliminated with the washing.4. This probably indicates that Ni²⁺ mainly inhibited the K⁺-induced phasic tension by reducing Ca²⁺ release rather than Ca²⁺ influx.     

Myometrial expression of small conductance Ca2+-activated K+ channels depresses phasic uterine contraction

Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specific isoform of small conductance Ca²⁺-activated K⁺ (SK) channel, SK3, promotes feedback regulation of myometrial Ca²⁺ and hence relaxation of the uterus. To determine the specific functional impact of SK3 channels, we assessed isometric contractions of uterine strips from genetically altered mice (SK3^T/T), in which SK3 is overexpressed and can be suppressed by oral administration of doxycycline (SK3^T/T+Dox). We found SK3 protein in mouse myometrium, and this expression was substantially higher in SK3^T/T mice and lower in SK3^T/T+Dox mice compared with wild-type (WT) controls. Sustained contractions elicited by 60 mM KCl were not different among SK3^T/T, SK3^T/T+Dox, and WT mice. However, the rate of onset and magnitude of spontaneously occurring phasic contractions was muted significantly in isolated uterine strips from SK3^T/T mice compared with those from WT mice. These spontaneous contractions were augmented greatly by blockade of SK channels with apamin or by suppression of SK3 expression. Phasic but not tonic contraction in response to oxytocin was depressed in uterine strips from SK3^T/T mice, whereas suppression of SK3 channel expression or treatment with apamin promoted the predominance of large coordinated phasic events over tone. Spontaneous contractions and the phasic component of oxytocin contractions were blocked by nifedipine but not by cyclopiazonic acid. Our findings suggest that SK3 channels play an important role in regulating uterine function by limiting influx through L-type Ca²⁺ channels and disrupting the development of concerted phasic contractile events. uterus; Ca²⁺-activated K⁺ channel; doxycycline; mouse     

Characterization of the tissue‐level Ca2+ signals in spontaneously contracting human myometrium

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio‐temporal properties of tissue‐level [Ca²⁺]_i transients in thin slices of intact human myometrium. We found that the waveform of [Ca²⁺]_i transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio‐temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca²⁺]_i transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long‐lasting [Ca²⁺]_i transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca²⁺]_i oscillations that did not trigger contractions. The [Ca²⁺]_i oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T‐type Ca²⁺ channel inhibitor NNC55‐0396. In conclusion, our data suggest that the spread of [Ca²⁺]_i signals in human myometrium is achieved via propagation of long‐lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

Background/Aims

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca²⁺ release from sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) -dependent intracellular Ca²⁺ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking.

Methodology/Principal Findings

Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K⁺ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca²⁺ stores, but were inhibited in low Ca²⁺ solution or in the presence of nifedipine, an inhibitor of L-type Ca²⁺channels. ICs were found in small numbers in the mouse cervix but not in the vagina.

Conclusions/Significance

Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca²⁺ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.     

Effects of noradrenaline on45Ca2+ efflux from isolated rat islets of Langerhans

Noradrenaline caused a prompt but transient increase in the rate of⁴⁵Ca²⁺ efflux from isolated rat islets of Langerhans perifused in Ca²⁺ depleted medium. The response was modest in size and was unaffected by isosmotic replacement of NaCl with choline chloride or by inclusion of 0.5 mM dibutyryl cAMP in the perifusion medium, suggesting that it was not mediated by Na+: Ca²⁺ exchange nor by lowered cAMP. Despite its effect on⁴⁵Ca²⁺ efflux, noradrenaline treatment did not alter the kinetics of⁴⁵Ca²⁺ efflux in response to the muscarinic agonist, carbamylcholine, nor did it change the magnitude of the response to this agent. Simultaneous introduction of 20 mM glucose with noradrenaline prevented a rise in⁴⁵Ca²⁺ efflux and indeed resulted in inhibition of⁴⁵Ca²⁺ efflux. The data suggest that noradrenaline does not directly activate the mechanisms which regulate Ca²⁺ extrusion from islets cells, and they do not support a primary role for the Ca²⁺ efflux response in mediating adrenergic inhibition of insulin secretion.     

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl₂, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca²⁺]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K⁺]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.     

Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor

To investigate the relationship between the oxytocin (OT) receptor (OTR) quantity and the contractile features systematically, we measured the mRNA expression levels of OTR and L-type Ca(2+) channel alpha(1C)-subunit (alpha(1C)) and examined the regulatory mechanisms of OT-induced phasic or tonic contractions of the longitudinal smooth muscles in mouse uteri. The mRNA expression of OTR in 19.0 G (19.0 days of gestation) was greater than those in nonpregnant phases, and that of alpha(1C) in estrus and 19.0 G was higher than in diestrus. OT-induced contractions sparsely occurred in diestrus. The OT-induced all-or-none-type phasic contractions at low concentrations were abolished by verapamil in both estrus and 19.0 G. OT-induced tonic contractions had similar pD(2) values in both estrus and 19.0 G. However, the magnitude in 19.0 G was much greater than that in estrus. The large tonic contractions also occurred in PGF(2alpha) receptor (FP) knockout mice in 19.0 G despite a small amount of OTR. Verapamil and Y-27632 partially inhibited the tonic contractions in 19.0 G. Cyclopiazonic acid-induced tonic contractions were reciprocally decreased with the increase in the OT-induced ones in 19.0 G. These results indicate that the phasic contractions are dependent on alpha(1C). The tonic contractions in 19.0 G are dependent on both Ca(2+) influxes via L-type Ca(2+) channels and store-operated Ca(2+) channels, and the force is augmented by the Rho signal pathway, which increases the Ca(2+) sensitivity. Thus the uterine contractions are mainly controlled by the modification of contractile signal machinery rather than simply by the OTR quantity.     

Carbachol- and KCl-induced changes in phosphoinositide metabolism and free calcium in guinea pig cerebral cortex synaptosomes

Phosphoinositide (PI) and calcium metabolism were studied in guinea pig cerebral cortex synaptosomes. Mass amounts of inositol and inositol monophosphates, and the levels of free intrasynaptosomal calcium ([Ca²⁺]_i) were measured after KCl (60 mM), after a direct cholinergic agonist carbachol (CA, 1mM), and after their combination. Inositol, inositol-1-phosphate (Ins1P), inositol-4-phosphate (Ins4P) and [Ca²⁺]_i were measured with and without 10 mM LiCl in the incubation medium. CA-induced cholinergic stimulation elevated synaptosomal Ins4P levels by 40% but did not affect Ins1P or [Ca²⁺]_i. On the contrary, KCl elevated Ins1P by 50% and [Ca²⁺]_i by 40% above the resting level, and decreased inositol by 20%, whereas no alterations in Ins4P occurred. CA did not modify the response of KCl, but KCl abolished the elevation of Ins4P by CA. LiCl attenuated KCl-induced elevation of Ins1P but amplified the CA-induced elevation of Ins4P. The elevation of presynaptic [Ca²⁺]_i was accompanied by accumulation of Ins1P but not that of Ins4P. Hence, the present results suggest that presynaptic cholinergic stimulation and KCl-induced depolarization may activate different degradation pathways of inositolphosphate metabolism.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca²⁺-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca²⁺-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of ⁴⁵Ca from cells preequilibrated with ⁴⁵Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular ⁴⁵Ca level. Inositol trisphosphate, but not cAMP, evoked ⁴⁵Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Effect of yttrium on calcium-dependent processes in vertebrate myocardium

Inoptopic effect of yttrium acetate (Y³⁺) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y³⁺ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y³⁺ induced inhibition of energy-dependent Ca²⁺ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH₄NO₃ and Ca²⁺ or 25 mM potassium acetate, 100 mM sucrose and Ca²⁺. It is suggested that the Y³⁺-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y³⁺ on potential-modulated Ca²⁺-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Ca²⁺-carrier in IMM. The data confirming that Y³⁺ activates energy-dependent K⁺ transport catalyzed by mitochondrial uniporter and blocks Ca²⁺-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y³⁺ action on vertebrates and human CM.     

Role of Rho-kinase in mediating contraction of chicken embryo femoral arteries

Rho-kinase-dependent Ca²⁺ sensitization is an essential process for contraction of mammalian vascular smooth muscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using the Rho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca²⁺ sensitization in depolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoral arteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of which were abolished in the absence of extracellular Ca²⁺. Hydroxyfasudil (10 μM) left the initial phasic component nearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractions elicited by phenylephrine (PE), the thromboxane A₂ mimetic U46619, and endothelin-1. In contrast, inhibition of protein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participation of PKC-mediated Ca²⁺ sensitization. Incubation under chronic hypoxia (15% O₂ from day 0) impaired embryonic growth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of a role for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries.     

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca²⁺]_i) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca²⁺]_i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca²⁺]_i after application at the doses of 30 nM (n?=?34, p?<?0.01), 100 nM (n?=?41, p?<?0.001) and 300 nM (n?=?46, p?<?0.001). The stimulatory effect of OT (300 nM) on [Ca²⁺]_i was persistent in Ca²⁺-free conditions (n?=?56, p?<?0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca²⁺]_i (n?=?28, p?<?0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.     

Phosphodiesterase types 3 and 4 regulate the phasic contraction of neonatal rat bladder smooth myocytes via distinct mechanisms

Activation of the cyclic AMP (cAMP) pathway reduces bladder contractility. However, the role of phosphodiesterase (PDE) families in regulating this function is poorly understood. Here, we compared the contractile function of the cAMP hydrolyzing PDEs in neonatal rat bladder smooth myocytes. RT-PCR and Western blotting analysis revealed that several isoforms of PDE1–4 were expressed in neonatal rat bladder. While 8-methoxymethyl-3-isobutyl-1-methylxanthine (a PDE1 inhibitor) and BAY-60-7550 (a PDE2 inhibitor) had no effect on the carbachol-enhanced phasic contractions of bladder strips, cilostamide (Cil, a PDE3 inhibitor) and Ro-20-1724 (Ro, a PDE4 inhibitor) significantly reduced these contractions. This inhibitory effect of Ro was blunted by the PKA inhibitor H-89, while the inhibitory effect of Cil was strongly attenuated by the PKG inhibitor KT 5823. Application of Ro in single bladder smooth myocytes resulted in an increase in Ca^2 + spark frequency but a decrease both in Ca^2 + transients and in sarcoplasmic reticulum (SR) Ca^2 + content. In contrast, Cil had no effect on these events. Furthermore, Ro-induced inhibition of the phasic contractions was significantly blocked by ryanodine and iberiotoxin. Taken together, PDE3 and PDE4 are the main PDE isoforms in maintaining the phasic contractions of bladder smooth myocytes, with PDE4 being functionally more active than PDE3. However, their roles are mediated through different mechanisms.     

Modulation of adrenal cell functions by cadmium salts. 4. Ca2+-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis

In previous studies, nonlethal CdCl₂ concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl₂ inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl₂. Since CdCl₂ competed at metabolic steps requiring Ca²⁺ in other tissues, experiments were designed to examine Cd²⁺ competition with Ca²⁺ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl₂ were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl₂ in the presence or absence of EGTA, a relatively specific Ca²⁺, but not Cd²⁺, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl₂, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca²⁺ transfer across plasma membranes. Besides determining Ca²⁺ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca²⁺ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl₂, ACTH, ACTH/CdCl₂ or ACTH followed after 50 s by CdCl₂. Using Ca²⁺-supplemented media, we observed that Cd²⁺ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca²⁺-containing medium supplemented with Ca²⁺ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd²⁺-treated cells was only reduced by EGTA, when Ca²⁺ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd²⁺-treated cells, suggesting that extracellular Ca²⁺ resources may compete against Cd²⁺ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca²⁺ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca²⁺ concentrations before returning to basal, steady-state levels. Cd²⁺ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd²⁺ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl₂-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd²⁺ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd²⁺-induced hyperbolic rise in intracellular Cd²⁺. These fluorescence measurements suggested that cytoplasmic Ca²⁺ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca²⁺ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd²⁺ freely enters the cell under basal conditions and Cd²⁺ entry is accelerated by ACTH stimulation. Data were consistent with Ca²⁺ being required for optimal stimulated steroid production and Cd²⁺ probably competing with Ca²⁺ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.