Intracellular Application Of GDPbetaS Research Articles

Multiple regulation by external ATP of nifedipine‐insensitive, high voltage‐activated Ca2+ current in guinea‐pig mesenteric terminal arteriole

We investigated the receptor-mediated regulation of nifedipine-insensitive, high voltage-activated Ca(2+) currents in guinea-pig terminal mesenteric arterioles (I(mVDCC)) using the whole-cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on I(mVDCC). The effects of ATP on I(mVDCC) after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mM Ba(2+), ATP potentiated I(mVDCC) at low concentrations (approximately 1-100 microM), but inhibited it at higher concentrations (>100 microM). The potentiating effects of ATP were abolished by suramin (100 microM) and PPADS (10 microM) and by intracellular application of GDPbetaS (500 microM), whereas a substantial part of I(mVDCC) inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation-free solution, I(mVDCC) was enlarged and underwent biphasic effects by ATPgammaS and ADP, while 2-methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP-induced I(mVDCC) potentiation was selectively inhibited by anti-Galpha(s) antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP-induced inhibition was selectively inhibited by Galpha(q/11) antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for I(mVDCC) potentiation with PKC inhibitors was: ATPgammaS > ATP>/=ADP and for inhibition with PKA inhibitors was: 2MeSATP > ATPgammaS > ATP > ADP. Neither I(mVDCC) potentiation nor inhibition showed voltage dependence. These results suggest that I(mVDCC) is multi-phasically regulated by external ATP via P2Y(11)-resembling receptor/G(s)/PKA pathway, P2Y(1)-like receptor/G(q/11)/PKC pathway, and metal chelation.

Intracellular Ca(2+) modulation of ATP-sensitive K(+) channel activity in acetylcholine-induced activation of rat pancreatic beta-cells.

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.

Changes of the responses of single sympathetic ganglionic neurones to substance P following desensitization

1. The neuropeptide substance P (SP) exerts an excitatory effect on sympathetic neurones by inhibiting a time- and voltage-dependent potassium current. During prolonged application of SP, the response desensitizes. The changes in kinetics of the SP response in single neurones after desensitization have been studied in an attempt to gain some insight as to the molecular mechanism of desensitization in live, functioning neurones. 2. Desensitization to SP resulted in subsequent SP responses being smaller, but the time course was unchanged in desensitized cells compared with non-desensitized cells. 3. Experimental manipulations were performed to decrease receptor and G protein function for comparison to desensitization. Intracellular application of GDPbetaS, to decrease G protein function, led to successive responses to agonist becoming smaller and slower. When functional muscarinic receptors were decreased by extracellular application of propylbenzilylcholine mustard (PrBCM), the response to muscarine became smaller, but the time course was unchanged compared with the change in time course produced by PrBCM vehicle alone. 4. The results have also been compared with simulations from a mathematical model of drug-receptor-G protein interactions. Under a constrained set of conditions, the model predicts that decreasing the size of the G protein pool will decrease both the magnitude and the time course of the response to agonist. Decreasing receptor levels results in a more efficient decrease in the magnitude of the response but no change in the time course of the response. 5. These data provide evidence that desensitization of the response to SP in single neurones results from a decrease in functional receptors.

Amphetamine elicited potential changes in vertebrate and invertebrate central neurons

The effects of amphetamine on potential changes in both vertebrate and invertebrate central neurons and factors affecting the potential changes were tested. The animals studied included mice, newborn rat and African snail. Seizure was elicited after lethal doses of d-amphetamine (75 mg/kg, i.p.) administration in mice. Repetitive firing of the action potentials were elicited after d-amphetamine (1-30 microM) administration in thin thalamic brain slices of newborn rat. Bursting firing of action potentials in the giant African central RP4 neuron were also elicited after d-amphetamine or l-amphetamine (0.27 mM) administration. The amphetamine elicited bursting firing of action potentials was not blocked even after high concentrations of d-tubocurarine, atropine, haloperidol, hexamethonium administration. Therefore, the amphetamine elicited potential changes may not be directly related to the activation of the receptors of the neuron. The bursting firing of action potentials elicited by amphetamine occurred 20-30 min after amphetamine administration extracellularly, even after high concentrations of d-amphetamine administration (0.27, 1 mM). However, the bursting firing of potentials occurred immediately if amphetamine was administrated intracellularly at lower concentration. Extracellular application of ruthenium red, the calcium antagonist, abolished the amphetamine elicited bursting firing of action potentials. If intracellular injection of EGTA, a calcium ion chelator, or injection with high concentrations of magnesium, the bursting firing of potentials were immediately abolished. These results suggested that the active site of amphetamine may be inside of the neuron and the calcium ion in the neuron played an important role on the bursting of potentials. In two-electrode voltage clamped RP4 neuron, amphetamine, at 0.27 mM, decreased the total inward and steady outward currents of the RP4 neuron. d-Amphetamine also decreased the calcium, Ia and the steady-state outward currents of the RP4 neuron. Besides, amphetamine elicited a negative slope resistance (NSR) if membrane potential was in the range of -50 to -10 mV. The NSR was decreased in cobalt substituted calcium free and sodium free solution. The effects of secondary messengers on the amphetamine elicited potential changes were tested. The bursting firing of action potentials elicited by amphetamine in central snail neurons decreased following extracellular application of H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after extracellular application of H7 (1,(5-isoquinolinesulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neuron may be associated with the cyclic AMP second messenger system; on the other hand, it may not be associated with the G protein and protein kinase C activity. It is concluded that amphetamine elicited potential changes in both vertebrate and invertebrate central neurons. The changes are closely related to the ionic currents and second messengers of the neurons.

Alpha2-Adrenoceptor-mediated potassium currents in acutely dissociated rat locus coeruleus neurones.

1. The noradrenaline (NA)-activated response was investigated in neurones acutely dissociated from the rat locus coeruleus (LC) using nystatin-perforated, conventional whole-cell and inside-out patch recording modes under current- and voltage-clamp conditions. 2. Under current-clamp conditions, NA hyperpolarized the LC neurones, abolishing the spontaneous action potentials. In voltage-clamp studies, NA induced an inwardly rectifying K+ current (INA) in a concentration-dependent manner with a half-maximum effective concentration of 2.2 x 10(-7) M. 3. INA was mimicked by the alpha2-agonist UK14304 but was inhibited by either the alpha2B/alpha2C antagonist ARC239 or the alpha1- and alpha2B/alpha2C antagonist prazosin, suggesting the contribution of alpha2B/alpha2C adrenoceptors. 4. INA was inhibited by the intracellular application of GDPbetaS but fully activated by intracellular perfusion of GTPgammaS. 5. In the inside-out recording mode, the application of GTP to the cytoplasmic side of the patch membrane markedly enhanced the open probability of the NA-activated single channels which represented the inwardly rectifying properties. 6. These results indicate that the activation of alpha2B/alpha2C adrenoceptors coupled with GTP-binding protein directly activates the inwardly rectifying K+ currents in rat LC neurones, thus resulting in a decrease in the spontaneous firing activities.

Substance P suppresses GABAA receptor function via protein kinase C in primary sensory neurones of bullfrogs.

1. The effects of substance P (SP) and related tachykinins on the function of gamma-aminobutyric acid-A (GABAA) receptors were examined in acutely dissociated neurones of bullfrog dorsal root ganglia (DRG) by using whole-cell voltage-clamp techniques. 2. Application of SP (10 nM to 1 microM) depressed inward currents produced by GABAA receptor activation (IGABA). Neurokinin A (NKA) and neurokinin B (NKB) also depressed IGABA; the rank order of agonist potency was SP > NKA > NKB. Spantide ([D-Arg1, D-Trp7,9,Leu11]SP) and L-703,606, NK1 receptor antagonists, blocked the SP-induced depression of IGABA. 3. SP irreversibly depressed IGABA, when neurones were intracellularly dialysed with GTP gamma S. Intracellular application of GDP beta S prevented the SP-induced depression of IGABA. Pertussis toxin (PTX) did not block the inhibitory effect of SP on IGABA. 4. The depression of IGABA produced by SP was inhibited by H-7 and PKC(19-36), protein kinase C (PKC) inhibitors, but not by H-9 and HA-1004, protein kinase A inhibitors. IGABA was suppressed by application of sn-1,2-dioctanoyl glycerol (DOG), a PKC activator. 5. It is concluded that activation of neurokinin-1 (NK1) receptors downregulates the function of the GABAA receptor of primary sensory neurones through a PTX-insensitive G-protein. PKC may be involved in the transduction pathway of the tachykinin-induced inhibition of the GABAA receptor.

Alpha 1 adrenoceptor activation potentiates taurine response mediated by protein kinase C in substantia nigra neurons.

1. The potentiation of glycine receptor-mediated taurine response (Itau) by alpha 1 adrenoceptor activation was investigated in neurons freshly dissociated from the rat substantia nigra (SN) using a nystatin perforated-patch recording. 2. Norepinephrine (NE) at a concentration of 10(-4) M in the presence of 10(-5) M yohimbine and 10(-5) M propranolol potentiated the peak amplitude of Itau (10(-3) M) at a holding potential of -40 mV under voltage clamp conditions. NE could be substituted by phenylephrine at this potentiation. 3. This potentiation of the taurine response persisted in the treatment with pertussis toxin (500 ng/ml) for 18 h. The intracellular application of GDP-beta S (100 microM) with a conventional whole cell patch recording mode abolished the effect of alpha 1 adrenoceptor activation on the Itau. 4. Staurosporine (10(-7) M) blocked the enhancement of Itau by 10(-4) M NE with 10(-5) M yohimbine and 10(-5) M propranolol. In additional phorbol-12-myristate 13-acetate (10(-5) M) potentiated Itau. 5. The intracellular application of 0.275 U/ml protein kinase C (PKC) with a conventional whole cell configuration gradually increased the peak amplitude of Itau. On the other hand, intracellular perfusion either without PKC or with PKC plus 4 microM PKC (19-36), a PKC inhibitor, did not potentiate Itau. 6. A single channel recording in a cell attached configuration revealed that NE (10(-4) M) with 10(-5) M yohimbine and 10(-5) M propranolol increased the total open time of the taurine-activated channel. This increase of the channel opening was antagonized by staurosporine (10(-7) M). 7. Neither tapsigargin (10(-6) M), LiCl (10(-4) M), trifluoperazine (10(-5) M) nor (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (10(-4) M) applied in the perfusate were found to affect the potentiation of Itau by alpha 1 adrenoceptor. The intracellular application of inositol triphosphates (10(-4) M) in a conventional whole cell recording also had no effect on Itau. 8. These findings thus indicate that alpha 1 adrenoceptor coupled with pertussis-insensitive G protein increases the intracellular PKC activity, thus leading to an increase in the channel opening activated by taurine and an enhancement of the peak amplitude of Itau in the SN neurons.

Facilitation of T-type calcium current in bullfrog atrial cells: voltage-dependent relief of a G protein inhibitory tone.

1. The properties of the low-threshold calcium current, ICa,T, were investigated in bullfrog isolated atrial cardiomyocytes using the whole-cell, patch-clamp technique under control conditions and during beta-adrenergic stimulation. 2. The intracellular application of GTP gamma S or adenosine-5'-O-3-thiotriphosphate (ATP gamma S), poorly hydrolysable analogues of GTP and ATP, respectively, barely affected ICa,T amplitude in control conditions. beta-Adrenergic stimulation effects were more marked in the presence of ATP gamma S. 3. The intracellular application of GDP beta S and ADP reduced ICa,T amplitude. In cells pretreated with pertussis toxin, ICa,T amplitude was significantly increased. In both conditions, the addition of isoprenaline was without effect. 4. Under both control and beta-adrenergic-stimulated conditions, a conditioning prepulse to +70 mV did not fully inactivate ICa,T; rather ICa,T facilitation often occurred after beta-adrenergic stimulation. 5. In GTP gamma S- and ATP gamma S-dialysed cells, ICa,T facilitation was generally observed after a prepulse; it was larger in the ATP gamma S dialysis. Facilitation was sustained but ended immediately upon cessation of conditioning prepulses. After beta-adrenergic stimulation, facilitation was more marked in GTP gamma S- than in ATP gamma S-dialysed cells. 6. ICa,T facilitation was prevented by the intracellular application of GDP beta S and by pertussis toxin pretreatment. 7. ICa,T facilitation developed markedly in the presence of intracellular cyclic AMP. This effect was prevented by pertussis toxin pretreatment of the cells. 8. It is thus proposed that ICa,T is under a double antagonistic control by both a Gs and a Gi protein. Furthermore, the double-pulse-induced facilitation of ICa,T results from a voltage-dependent relief of the Gi protein inhibitory tone. Such an effect is increased by protein kinase A-dependent phosphorylation, presumably of the Gi protein.

Inhibition of inwardly rectifying K+ current by external Ca2+ ions in freshly isolated rabbit osteoclasts.

1. Regulation of membrane potential by extracellular Ca2+ concentration ([Ca2+]o) was examined in freshly isolated rabbit osteoclasts. 2. The resting membrane potential of osteoclasts was close to the K+ equilibrium potential in 1 mM Ca2+ medium. An elevation of [Ca2+]o caused membrane depolarization, accompanied by a decrease in the membrane conductance. 3. The inwardly rectifying K+ current observed under voltage clamp was dose-dependently inhibited by an elevation of [Ca2+]o, which explained the membrane depolarization caused by high [Ca2+]o. 4. Other divalent cations also inhibited the inwardly rectifying K+ current with the following order of potency: Ca2+ < Ni2+ < or = Co2+ < Cd2+. 5. In the presence of intracellular GTP gamma S the inwardly rectifying K+ current was irreversibly inhibited by [Ca2+]o, whereas the inhibition of the inwardly rectifying K+ current was greatly attenuated by intracellular application of GDP beta S. 6. Pertussis toxin (PTX) treatment did not abolish the inhibition of the inwardly rectifying K+ current caused by [Ca2+]o. 7. These results suggest that inwardly rectifying K+ channels in osteoclasts were regulated by a PTX-insensitive G-protein, which was coupled to the putative Ca2+ receptor or sensor on the cell membrane.

Dopamine neuron membrane physiology: Characterization of the transient outward current (IA) and demonstration of a common signal transduction pathway for IA and IK

Dopamine neurons derived from the mesencephalon of embryonic rats were maintained in primary culture, identified and studied with whole-cell patch recording techniques. These neurons demonstrated a rapidly activating and inactivating voltage-dependent outward current which required the presence of K+ ions. This current was termed IA because of its transient nature. It was elicited by step depolarizations from holding potentials more negative than -50 mV and exhibited steady-state inactivation at a membrane potential more positive than -40 mV and half-maximal inactivation observed at -65 mV. This current rapidly achieved peak activation in less than 8 msec and decayed with a time constant (tau) of 58 +/- 5 msec. This current was observed in the presence of tetraethylammonium but was readily blocked by 4-aminopyridine (2-4 mM). This current was also observed to be modulated by stimulation of D2 dopamine receptors (DA autoreceptors) located on the dopamine neurons. Thus, both DA and the D2 receptor agonist quinpirole enhanced the peak IA observed, while the partial D1 receptor agonist SKF 38393 was without effect. The enhancement of IA was confirmed to be due to the activation of D2 receptors as the effects of either DA or quinpirole were blocked by the D2 receptor antagonists eticlopride and sulpiride, but not by the D1 receptor antagonist SCH 23390. Since we have previously demonstrated that the IK present in these cells is also enhanced by D2 receptor stimulation, we investigated the signal transduction pathways involved in coupling DA autoreceptors to both IA and IK. The response of both these potassium currents to DA autoreceptor stimulation was completely abolished by the preincubation of cultures with pertussis toxin, indicating the possible involvement of the G proteins Gi and G(o). In an attempt to further characterize which G protein may be involved, additional experiments were performed. The ability of DA autoreceptor stimulation to augment both currents was also blocked completely when G protein activation was prevented by the intracellular application of GDP beta S (100 microM). In contrast, irreversible activation of G proteins by intracellular application of the nonhydrolyzable GTP analog GTP gamma S (100 microM) mimicked the effects of DA autoreceptor stimulation on both IA and IK. In addition, the intracellular application of a polyclonal antibody that was selective for the alpha-subunit of G(o) completely abolished the DA autoreceptor modulation of both currents while preimmune serum was without effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol trisphosphate-dependent mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) that appear to be independent of cAMP. To study the mechanisms involved in this response, we used the patch-clamp technique to microperfuse various agents into single rat gonadotrophs while monitoring [Ca2+]i with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (> 1 microM) oscillations in [Ca2+]i, which were blocked by intracellular application of GDP beta S (guanosine 5'-O-2-thiodiphosphate), indicating the involvement of a G-protein. To identify the intracellular messenger(s) involved, we microperfused gonadotrophs with cAMP, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of cAMP (100 microM) had no significant effect on basal [Ca2+]i and did not alter the PACAP-stimulated Ca2+ response. Heparin, but not its inactive isoform, completely blocked the PACAP-stimulated increase in [Ca2+]i, while Ins(1,4,5)P3 stimulated oscillations in [Ca2+]i very similar to those observed in response to PACAP. These results strongly suggest that PACAP mobilizes Ca2+ through an Ins(1,4,5)P3-dependent mechanism. The fact that PACAP stimulates two signaling pathways in pituitary cells could substantially enhance the signaling potential of this hypothalamic peptide.