Time Course Of Desensitization Research Articles

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices.

1. Responses in brain produced by the activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [3H]-noradrenaline from prisms of rat hippocampus. 2. AMPA (1-1000 microM) stimulated the release of [3H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 microM), LY 293558 (1 and 10 microM) and GYKI 52466 (10 and 30 microM). 3. By use of an experimental procedure with consecutive applications of AMPA (100 microM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1-300 microM). The concentration-response curve for AMPA-induced release of [3H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus. 4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 microM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was longlasting (second application of cyclothiazide was ineffective). 5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [3H]-noradrenaline: forskolin (10 microM, +78%), Rp-cAMPS (100 microM, +65%), Ro 31-8220 (10 microM, +163%) and thapsigargin (100 pM, + 161%). 6. The AMPA receptor-mediated response regulating the release of [3H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca2+ stores.

Rapid drug application resolves two types of nicotinic receptors on rat sympathetic ganglion cells.

The properties of nicotinic acetylcholine receptors (AChRs) on cultured rat superior cervical ganglion (SCG) neurons were analysed. AChR agonists [1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), cytisine] were applied to whole cells within 70ms. The desensitization rate of whole-cell currents during constant application of DMPP varied between neurons. The time course of desensitization was fitted by double exponentials with time constants kfast, of between 0.35 and 0.55s, and kslow, of 3-5s. By exchanging intracellular chloride for caesium methanesulphonate, the possibility of interference by a calcium-activated chloride current was excluded. In cells that exhibited a slowly desensitizing current during the application 20 microM DMPP, equimolar cytisine induced a larger peak current compared to the response to DMPP, while in cells with rapidly desensitizing DMPP-induced currents the response to equimolar cytisine was smaller. The differences in desensitization rates and agonist potencies are due to different functional properties of AChR subtypes, as indicated by currents recorded from outside-out patches upon rapid agonist application and removal (2ms each). The results indicate the presence of two distinct AChR subtypes on SCG neurons: one with a fast and one with a slow activation/desensitization rate, but both with similar single-channel conductances. Slow activation/desensitization was found to be associated with a high potency of cytisine/low potency of DMPP. For AChRs with rapid activation/desensitization kinetics the agonist potencies were reversed.

Distribution of desensitization time constants of mouse embryonic-like nicotinic and homomeric GLUR6 glutamate receptor channels

The distribution of the desensitization time constant, τ D, of two different, molecular well defined ligand gated receptor channels was analyzed: the embryonic-like nicotinic receptor (nAChR) channel of cultured mouse myotubes and recombinant, homomeric GluR6 receptor channels transiently expressed in HEK293 cells. Experiments were performed using outside-out patches in combination with a system for fast application of agonists which allows solution exchange within 0.1 ms. In response to application of saturating concentrations of acetylcholine (ACh) or glutamate (Glu), the peak current was reached in a submillisecond range and decayed monexponentially in the presence of the agonist, due to desensitization. τ D varied from 10 ms to 100 ms with a mean value of 55.0±22.6 ms ( n=133) in response to pulses of 10 −4 M ACh for embryonic-like nAChR channels and from 2.6 ms to 8.9 ms with a mean value of 5.0±1.9 ms ( n=35) in response to pulses of 10 −2 M Glu for homomeric GluR6 receptor channels. The reason for the high variability of the time course of desensitization is at present unclear.

Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3 gamma 2 than for alpha 1 beta 3 subunit receptors (0.13 vs. 0.03 s-1, respectively). The second phase of recovery for the two receptors were the same (approximately 0.003 s-1). 7. There was only a poor correlation between agonist potency and the degree or time course of desensitization. Isoguvacine (EC50 approximately to 10 microM) induced biphasic relaxation for both alpha 1 beta 3 and alpha 1 beta 3 gamma 2 subunit receptors (tau 1 = 288.6 +/- 43.3 and 167 +/- 15 ms, and tau 2 = 8.0 +/- 1.9 and 4.4 +/- 0.4 S, respectively, for each subunit combination). Taurine (EC50 approximately 7 mM) usually induced monophasic relaxation for both subunit combinations (tau 2 = 7.1 +/- 1.6 and 23.0 +/- 6.6 s, respectively). 8. A computer model was developed to examine the effect of the gamma 2 subunit on the time course of a synaptic potential. It was found that the gamma 2 subunit theoretically prolongs the time course of a synaptic potential by inducing desensitization more rapidly. The subsequent relaxation of the desensitized receptors through the open state increases Popen (the probability that the GABAA receptor is in an open conducting state) altering the time course of the modelled potential. alpha 1 beta 3 subunit receptors do not desensitize sufficiently rapidly to induce this desensitized state and, therefore, are shorter in time course. These data imply that the physiological role of the gamma 2 subunit is to increase synaptic efficacy by prolonging Popen.

Neuronal nicotinic acetylcholine receptors of ganglion cells in the cat retina.

Ganglion cells, retrogradely labeled with DiI, were dissociated from the cat retina. Under the whole-cell clamp mode using a patch pipette, ACh induced a transient current (IACh) in all cells tested. The soma diameter of ACh-responsive cells distributed from 11 to 23 microns, indicating that the preparation included Y, X, and W cells. IACh was inward rectifying. The reversal potential of IACh was close to the zero-current potential predicted from the intra- and extracellular monovalent cation concentrations by the Goldman-Hodgkin-Katz equation. The concentration-response curve gave a Hill coefficient of 0.89 and an EC50 of 3.2 x 10(-5) M. Nicotine (10(-4) M) induced a transient current, while muscarine (10(-4) M) was ineffective. The nicotinic antagonist, hexamethonium, reduced the amplitude of IACh in a concentration-dependent manner with an IC50 of 1.3 x 10(-6) M. The muscarinic antagonist, atropine, showed a concentration-dependent reduction in the amplitude of IACh with an IC50 of 4.4 x 10(-6) M. The amplitude of IACh was dependent on the extracellular Ca2+ concentration ([Ca2+]O); small at low [Ca2+]O and large at high [CA2+]O. The time course of desensitization was also dependent on [Ca2+]O; fast at low [Ca2+]O and slow at high [Ca2+]O. The replacement of extracellular Ca2+ to Sr2+ increased the amplitude of IACh, but replacement to other divalent cations reduced it. The results indicate the presence of functional neuronal nicotinic ACh receptors in Y, X, and W cells.

オピオイドレセプターの情報伝達系による脱感作の違い

RNA-injected Xenopus oocytes were used for studying homologous desensitization of opioid receptors to G-protein-coupled intracellular signaling pathways. In the oocytes expressing K opioid receptor, voltage-dependent Ca2+ channel α2+ and β subunits, the κ agonist U50488H (1 μM) inhibited Ca2+ channel current in a reversible manner, and the inhibition was diminished by sustained agonist exposure (homologous desensitization). More than 10 min was required to halve the inhibition of BI- or Q-type Ca2+ channels by the κ agonist, however, the t½ for U50488H-induced inhibition of BIII- or N-type channels was only 6±1 min. Moreover, in the oocytes expressing κ opioid receptor, EP4 prostaglandin receptor and CFTR channel, no apparent desensitization was observed in the κ receptor-mediated potentiation of the cyclic AMP production by prostaglandin E2 even when the oocytes were treated with U50488H for 2 hours or more. The time-course of desensitization of μ opioid receptor was also different in the effector molecules. These observations suggest that rate of homologous desensitization of opioid receptor is not only dependent on the receptor molecule itself but also on its intracellular signaling systems.

NMDA receptor channels: Subunit-specific potentiation by reducing agents

Sulfhydryl redox agents affect NMDA receptor activity. We investigated a putative redox site in four recombinant NMDA receptors. In 293 cells expressing NR1-NR2A channels dithiothreitol (DTT) rapidly potentiated l-glutamate-activated whole-cell currents and decreased the time course of desensitization and deactivation. Part of the current potentiation (reversible component) and all kinetic changes reversed upon washout. The remaining potentiation (persistent component) was abolished by an oxidizing agent. The N-terminal 370 residues of NR2A mediate the reversible component in chimeric N R2 subunits. I n cells expressing the NR1-NR2B, -NR2C, and -NR2D channels DTT elicited only a slowly developing, persistent potentiation and increased the deactivation time course. In these, but not in NR1-NR2A, the DTT effect was rendered insensitive to reoxidation by alkylation. Reduced glutathione mimicked the DTT effects only in the NR1-NR2A receptor. Hence, molecularly distinct NMDA receptors differ profoundly in their responses to sulfhydryl redox agents.

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle and the intracellular signal transduction

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle of the digestive tract is discussed together with the changes in intracellular signal transduction. Isolated single cells that show an all-or-none contractile response to acetylcholine were desensitized by treatment with 0.1 mM acetylcholine for 10 min, resulting in an increase in the threshold concentration of acetylcholine for contraction, but without changing any of the binding characteristics. Permeabilized cells showed that the desensitization is via uncoupling between the receptor and G-protein. Secretory cells (rat basophil leukemia-2H3 cells) transfected with human m3-receptor showed desensitization when treated with 0.1 mM carbachol for 30 min. The coupling between the receptor and G-protein was not impaired, but some unknown Ca(2+)-independent mechanism may be involved. Smooth muscle tissue was tested for its time-course of desensitization, and a novel transient resensitization was found at 1 min of incubation with 0.1 mM carbachol. This resensitization, and the desensitization prior to it, were accompanied with changes in binding affinity. However, the affinity was not changed, in parallel with desensitization afterwards, but the positive feedback loop of Ca(2+)-influx caused by alkalization via receptor-stimulation was suppressed. After a 30-min treatment, a Ca(2+)-independent mechanism caused the uncoupling and affinity decrease. Treatment for 3 hr increased the number of binding sites without recovery of the response. The desensitizing process is very diverse to achieve selectivity, but its purpose is in unity.

Peptide modulation of ACh receptor desensitization controls neurotransmitter release from chicken sympathetic neurons.

1. The distribution and release of substance P (SP) in embryonic chicken lumbar sympathetic ganglia was examined with the use of immunohistochemistry and radioimmunoassay, respectively. SP immunoreactivity was detected in nerve fibers surrounding individual sympathetic neurons and was released by ganglionic depolarization. 2. Effects of SP on nicotinic acetylcholine receptor (AChR) function was assayed in embryonic sympathetic neurons in vitro by whole-cell patch clamp. SP (0.1-20 microM) accelerated the rate of decay (desensitization) of ACh-induced currents. The AChR desensitization time course is biphasic and described by the sum of two exponential functions dependent on agonist concentration (time constant of the faster component, tau f = 1-2 s, and the slower time constant, tau s = 10-25 s). SP selectively decreased tau s and the contribution of the slow component to the overall rate of current decay. The effects of SP on desensitization were concentration dependent and reversible. SP slowed recovery from desensitization by 2.5-fold. 3. SP shifted the dose-response curve for ACh-induced desensitization, reducing the concentration of ACh required to produce half-maximal desensitization by approximately twofold. 4. Preapplication of SP was equivalent to SP applied together with ACh in accelerating AChR desensitization. SP did not alter the time course of currents elicited by nondesensitizing concentrations of ACh, carbamylcholine (CARB), or dimethylphenylpiperazinium (DMPP). These data suggest that AChR activation is neither necessary nor sufficient for the peptide to modulate receptor function. A kinetic model of the effects of SP on specific steps in AChR desensitization is presented. 5. SP enhanced the rate of decay of synaptic currents in sympathetic neurons innervated in vitro, decreasing the synaptic current duration by up to 80%. 6. Effects of SP on neurotransmitter release from sympathetic neurons were evaluated by measuring the release of [3H]-norepinephrine (NE). ACh and CARB stimulated NE release in a concentration- and calcium-dependent manner. SP alone had no effect on NE secretion, but the peptide inhibited NE release induced by ACh or CARB by 40-50%. 7. Although agonists specific for either nicotinic or muscarinic receptors stimulated release of NE, SP selectively inhibited the nicotinic component of transmitter secretion. Thus SP suppressed NE release induced by DMPP by up to 80% but had no effect on muscarine or depolarization-induced NE secretion. 8. Parallel studies of the modulatory effects of SP on whole-cell currents and NE secretion revealed that SP inhibition of transmitter release from sympathetic neurons is directly proportional to the extent of potentiation of AChR desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Homologous Desensitization of the Endothelin-1 Receptor Mediated Phosphoinositide Response in Cultured Neonatal Rat Cardiomyocytes

The goal of the present study was to identify the molecular mechanism underlying desensitization of endothelin-1 receptor-mediated phosphoinositide response in cultured neonatal rat heart cells. Endothelin elicited a concentration-dependent (EC 50=2.2 × 10 -9 M) increase of inositolphosphate production with a much higher potency then phenylephrine (EC 50=1.4 × 10 -6 M). Endothelin-1 (10 -8 M) evoked phosphoinositide turnover in the presence of 10 mM LiCl, which was greatly attenuated after 30-45 min of continuous stimulation with agonist, apparently resulting in a total absence of further inositolphosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li + was only present during the last 30 min of 150 min incubation, the inositolphosphate accumulation was decreased to a steady state of 33% of the initial rate. The loss of responsiveness of cardiomyocytes to endothelin-1 was not brought about by a limiting supply of phospholipase C substrate phosphatidylinositol 4,5-bishosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10 -8M endothelin-1 while the accumulation of inositolphosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10 -9M endothelin-1 the activation of phospholipase C by a second higher dose (10 -8 M) was severely (67%) inhibited at the same time leaving the induction of phosphoinositide turnover by phenylephrine (10 -4 M) virtually intact. Preincubation with phenylephrine (3 × 10 -6 M) also led to inhibition of the phenylephrine (10 -4 M) mediated inositolphosphate response (36% inhibition) while the endothelin-1 (10 -8 M) response was not affected. Addition of a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, led to inhibition of the endothelin-1 evoked phosphoinositide turnover but the rate of desensitization was not affected. Inhibition of protein kinase C with staurosporine did not alter the time course of desensitization. In conclusion, the activity of the phosphoinositide cycle in cardiomyocytes is homologously desensitized after stimulation with endothelin-1. The desensitization is not likely to be due to either depletion of phospholipase C substrate or to the activation of protein kinase C by inositol 1,4,5-trisphosphate-mobilized Ca 2+ and elevated 1,2-diacylglycerol levels.

Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C

In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity, are potent mitogens for CCL39 cells but were unable to stimulate either PLD or PC-PLC activity. Furthermore, exogenous addition of purified PC-PLC enzyme, although able to induce a strong and lasting hydrolysis of PC, was unable to produce a mitogenic signal on its own. On the basis of these results, we conclude that the activation of both PLD and PC-PLC is neither sufficient nor required to produce a mitogenic response.

Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells.

We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.

5-HT3 receptor channels in dissociated rat superior cervical ganglion neurons.

1. Whole-cell and single-channel voltage-clamp techniques were used to record the 5-HT3 receptor-mediated currents in neurons freshly dissociated from rat superior cervical ganglia. 2. Whole-cell currents elicited by brief pressure ejection of 5-HT (10 microM) reversed at -4.5 mV when extracellular and intracellular solutions mainly contained NaCl and CsCl. The peak current-voltage relation showed modest inward rectification that was fully developed within less than 2 ms of the applied voltage step. 3. With prolonged application of 5-HT (10 microM) using a fast perfusion system, the response desensitized in two phases with fast and slow time constants of 0.57 and 6.0 s at -74 mV. The time constants showed little voltage dependence; however, the relative amplitude of the two components was significantly dependent on voltage. The time course of desensitization was not affected by agents that increase the levels of intracellular cyclic AMP. 4. The relative permeability of the channel was determined from reversal potential changes. The channel passed small cations non-selectively, with permeability ratios (PX/PNa) of 0.93 and 1.24 for Cs+ and K+. The organic cations Tris and glucosamine were measurably permeant with permeability ratios of 0.19 and 0.06. Ca2+ was fairly permeant with a relative permeability of 0.55 in 20 mM solution and of 0.16 when the concentration of CaCl2 was increased to 115 mM. No permeability was detected for Cl-. 5. Fluctuation analysis of the whole-cell current revealed an apparent single-channel current of approximately 0.18 pA at -74 mV. 6. 5-HT-activated single-channel currents were recorded in excised outside-out patches. When 5-HT (10 microM) was delivered by pressure ejection, channel openings appeared rapidly with a delay of 28 ms. The unitary current was about approximately 0.80 pA at -74 mV. The channel activity induced by bath perfusion of 5-HT (0.8 microM) was significantly reduced by 100 nM of the 5-HT3 receptor-specific antagonists 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) or 3-tropanyl-indole-3-carboxylate (ICS 205-930). 7. The single-channel current-voltage relation was non-linear, with moderate inward rectification similar to that of the whole-cell current. The chord conductance of the channel decreased with membrane depolarization from 14.6 pS at -104 mV to only 9.9 pS at -54 mV. Open-time distributions consisted of two components with mean time constants of 0.45 and 2.8 ms at -104 mV. Burst-length distributions were also made up of two components with time constants of 0.45 and 4.6 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic agonist-induced actions on potassium and calcium channels in atrial myocytes: differential desensitization.

Desensitization to the effects of acetylcholine (ACh) or carbachol (CCh) on ACh-regulated potassium current (IK(ACh)) and the voltage-dependent L-type, calcium current (ICa(L)) was studied in single guinea-pig atrial cells under voltage clamp at room temperature (22-24 degrees C). At a holding potential of -40 mV, CCh (100 microM) activated IK(ACh) repeatedly and reproducibly when applied for 15-s periods with 60-s intervals between applications. Prolonged (10 min) exposure to CCh caused a time-dependent decline of IK(ACh) and a marked reduction (desensitization) of the response to subsequent application of CCh. In the absence of guanosine triphosphate (GTP) in the pipette, only partial resensitization occurred within 20 min after CCh washout. The desensitization to CCh could also be obtained when adenosine (20 microM) was used to activate this current. Therefore, this desensitization must be heterologous. In the presence of isoproterenol (ISO, 3 microM), ICa(L) increased. Acetylcholine continued to inhibit this current at a time when its activation of IK(ACh) had become desensitized. Muscarinic inhibition of ICa(L) eventually desensitized but only after IK(ACh) desensitization had occurred. Because of its heterologous nature, as well as of the ability of ACh to desensitize in the presence of intrapipette dextran, which is reported to inhibit beta-adrenergic receptor kinase (beta ARK), it seems unlikely that beta ARK-dependent phosphorylation of the muscarinic receptor (mAChR) accounted for desensitization. The differential desensitization time course is consistent with the hypothesis that different subunits of Gi, the inhibitory guanine nucleotide binding protein, not only transduce the agonist effects of ACh on IK(ACh) and ICa(L) but are also subjected to different inactivation/regulation processes.

? subunits determine the time course of desensitization in rat ?3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of alpha 3 beta 4 or alpha 3 beta 2 mRNAs. Currents of several microA amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n greater than or equal to 1.7) with a half-maximal activation concentration (EC50) of 15-30 microM. In alpha 3 beta 4 receptors, ACh displayed cooperativity (n = 1.8) but was less efficacious than DMPP, yet its EC50 was about equal to that of DMPP. Finally, in alpha 3 beta 2 receptors, ACh was much less efficacious and had a much lower EC50. Desensitization induced by either DMPP or ACh was slow in alpha 3 beta 4 nicotinic ACh receptors but was rapid and extensive in alpha 3 beta 2 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.

Activation and desensitization of the 5-HT3 receptor in a rat glioma x mouse neuroblastoma hybrid cell.

1. Tight-seal voltage-clamp techniques were used to study the 5-HT3 receptor of differentiated NG108-15 cells. 2. The inward current caused by 5-HT was dependent on the 5-HT concentration: the apparent dissociation constant was 3.3 microM and the Hill coefficient was 1.8. 3. Immediately after establishing a recording, sustained application of a saturating concentration of 5-HT caused the response to decline with a half-time of 0.57 s (at a membrane potential of -70 mV). The time course of desensitization was best fitted by a sum of two exponentials. 4. Desensitization became slower during the first 10 min of recording in the whole-cell configuration, with the half-time for response decay increasing to 1.8 s. The deceleration of desensitization may result from wash-out of a cytoplasmic regulator of the receptor. 5. Desensitization declined less during whole-cell recordings when patch pipettes contained non-hydrolysable analogues of adenosine 5'-triphosphate. 6. Desensitization developed more rapidly following the addition of forskolin, prostaglandin E1, cholera toxin or 1,9-dideoxyforskolin to the recording medium. Non-hydrolysable adenosine 5'-phosphate analogues had no effect on the enhancement of desensitization induced by forskolin.

Somatostatin inhibits the nicotinic receptor-activated inward current in guinea pig chromaffin cells

At a holding potential of −60 mV, bath application of somatostatin produced no current response itself, but inhibited the nicotinic inward current, a concentration of about 0.01 μM being required for a half-maximal effect. This inhibition was affected neither by pretreatment with somatostatin nor by injection of guanosine-5′-0-(3-thiotriphosphate) through a pathch pipette. When somatostatin (0.03–0.1 μM) produced its maximum inhibition, the proportion of plateau to peak nicotinic current was greatly reduced (to 5.3±5.7 % from the control value of 34.9±4.9 %, n=5) with a facilitation of the time course of desensitization. After washout of somatostatin, the nicotinic current was restored to control level in a time-dependent, rather than an activation-dependent manner. The results suggest that somatostatin inhibits the function of the nicotinic receptor by facilitation of its desensitization or by an open-channel block.

The properties of the ATP‐induced depolarization and current in single cells isolated from the guinea‐pig urinary bladder

1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0.5-1 microM). 3. ATP activated a dose-dependent inward current with a short latency (18 ms with 10 microM ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis-Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant (Kd) of 2.3 microM. The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials. 4. The reversal potential of the ATP-activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels. 5. alpha,beta-Methylene ATP also produced a dose-dependent inward current but was less potent than ATP (n: 1.6, Kd: 10.4 microM). alpha,beta-Methylene ATP blocked the response to ATP by desensitization of the receptor. 6. alpha,beta-Methylene ATP was 50-100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle. 7. The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.

Temperature‐dependence of desensitization induced by acetylcholine and histamine in guinea‐pig ileal longitudinal muscle

1. The effects of temperature on the time course of desensitization induced by acetylcholine and histamine, and on the recovery from desensitization were studied in the longitudinal muscle of the guinea-pig ileum. 2. Self- and cross-desensitization produced by acetylcholine (10(-5) M) occurred rapidly in the first 10 min of exposure to the agonist, with the same time course and the same degree of desensitization over the temperature range of 11 degrees C to 31 degrees C. 3. Self-desensitization produced by histamine (10(-5) M) also occurred rapidly in the first 10 min of exposure to the agonist, and showed great temperature-dependence, especially at 11 degrees C and 21 degrees C, but scarcely occurred at 6 degrees C. 4. Cross-desensitization produced by histamine developed gradually with time and showed a moderate temperature-dependence between 11 degrees C and 31 degrees C, but scarcely occurred at 6 degrees C. 5. The recovery processes from desensitization showed marked temperature-dependence. Recovery was halted completely at 11 degrees C. 6. These studies suggest that acetylcholine-induced desensitization may be attributed to a single non-specific mechanism. Histamine-induced desensitization may be due to at least two mechanisms: it occurs in both a specific and non-specific manner. Each of these desensitizations can be characterized by its unique temperature-dependence.

Glycine decreases desensitization of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes and is required for NMDA responses.

In Xenopus oocytes injected with rat brain mRNA, as in neurons, glycine greatly potentiated responses of the N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. Injected oocytes generated a partially desensitizing inward current in response to NMDA with 30 nM added glycine. As the added glycine concentration was increased from 30 nM to 1 microM, the NMDA response was increased and exhibited less desensitization. The relationship between the NMDA peak response and added glycine concentration indicated a single component response with apparent affinity of 0.29 microM and a Hill coefficient of 0.77. The desensitized response was also fit by the Hill relation with a lower affinity but similar coefficient. The time course of desensitization at 500 microM NMDA was exponential with a time constant (350 msec) that was independent of glycine concentration between 0.03 and 0.3 microM. At higher glycine concentration a slower component of decay (tau = 1.4 sec) was observed. This component was enhanced by increasing the extracellular Ca2+. NMDA without added glycine evoked a small transient response. However this response was suppressed completely by prewashing with the glycine antagonist 7-chlorokynurenic acid, suggesting that it may have been due to glycine contamination. The dose-response relation for low concentrations of glycine indicated that the measured level of glycine contamination accounted for these responses. These results indicate that glycine has at least two actions at the NMDA receptor: it enables channel opening by the agonist and decreases desensitization.