A1 Adenosine Receptor-Mediated Inhibition of Parasympathetic Neuromuscular Transmission in Human and Murine Urinary Bladder.

The potential role of A1 adenosine receptors in modulating neuromuscular transmission in the detrusor muscle of the urinary bladder has been tested in human and murine preparations with the intent to determine the viability of using adenosine receptor agonists as adjuncts to treat overactive bladder. In human detrusor muscle preparations, contractile responses to electrical field stimulation were inhibited by the selective A1 adenosine receptor agonists 2-chloro-N(6)-cyclopentyladenosine, N(6)-cyclopentyladenosine (CPA), and adenosine (rank order of potency: 2-chloro-N(6)-cyclopentyladenosine > CPA > adenosine). Pretreatment with 8-cyclopentyl-3-[3-[[4(fluorosulphonyl)benzoyl]oxy]propyl]-1-propylxanthine, an irreversible A1 antagonist, blocked the effects of CPA, thus confirming the role of A1 receptors in human detrusor preparations. In murine detrusor muscle preparations, contractions evoked by electrical field stimulation were reduced by CPA or adenosine. Amplitudes of the P2X purinoceptor-mediated excitatory junctional potentials (EJPs) recorded with intracellular microelectrodes were reduced in amplitude by CPA and adenosine with no effect on the spontaneous EJP amplitudes, confirming the prejunctional action of these agents. 8-Cyclopentyltheophylline, a selective A1 receptor antagonist, reversed the effects of CPA on EJP amplitudes with no effect of spontaneous EJPs, confirming the role of A1 receptors in mediating these effects.

Low-frequency neuromuscular depression is a consequence of a reduction in nerve terminal Ca2+ currents at mammalian motor nerve endings.

BACKGROUND: The decline in voluntary muscle contraction during low-frequency nerve stimulation is used clinically to assess the type and degree of neuromuscular block. The mechanism underlying this depression is unknown. METHODS: Simultaneous electrophysiological measurements of neurotransmitter release and prejunctional Ca currents were made at mouse neuromuscular junctions to evaluate the hypothesis that decreases in nerve terminal Ca currents are responsible for low-frequency depression. RESULTS: Under conditions generally used to measure Ca currents at the neuromuscular junction, increasing the frequency of nerve stimulation briefly from 0.017 to 0.1-1 Hz caused a simultaneous reduction in the release of the neurotransmitter acetylcholine to 52.2 ± 4.4% of control and the Ca current peak to 75.4 ± 2.0% of control (P < 0.001, n = 5 experiments for both measurements, mean ± SEM for all data). In conditions used for train-of-four monitoring (4 stimuli, 2 Hz), neurotransmitter release declined to 42.0 ± 1.0% of control and the Ca current peak declined to 75.8 ± 3.3% of control between the first and fourth stimulus (P < 0.001, n = 7 experiments for both measurements). Depression in acetylcholine release during train-of-four protocols also occurred in the absence of neuromuscular-blocking drugs. DISCUSSION: The results demonstrate that neuromuscular depression during train-of-four monitoring is due to a decline in nerve terminal Ca currents, hence reducing the release of acetylcholine. As similar processes may come into play at higher stimulation frequencies, agents that antagonize the decline in Ca currents could be used to treat conditions in which neuromuscular depression can be debilitating.

Selective disruption of the mammalian secretory apparatus enhances or eliminates calcium current modulation in nerve endings.

Modulation of secretion via G protein-coupled receptors (GPCRs) serves an important regulatory function in neuronal and nonneuronal secretory cells. Most secretory cells possess voltage-gated calcium channels, share homologues of the core complex of three proteins (the SNAREs) that constitute the secretory apparatus, and are modulated by GPCR activation. Activators of GPCRs generally inhibit the release of neurotransmitter substances to a maximum of only 50-60% of the control level, suggesting that complex protein-protein interactions may govern the efficacy of this form of modulation. In this article, molecular genetic approaches are used in combination with botulinum toxins (selective molecular scalpels that cleave the SNAREs at highly restricted loci) to address this issue. The results suggest that the cleavage of either of the plasma membrane SNAREs (syntaxin or SNAP-25) prevents modulation of calcium currents by A(1) adenosine receptors at mammalian motor nerve endings. In contrast, cleavage of the synaptic vesicle SNARE (synaptobrevin) in conjunction with deletion of the vesicle-docking protein Rab3A greatly enhances the efficacy of calcium current modulation.

Modulation of calcium-dependent and -independent acetylcholine release from motor nerve endings.

Inhibition of acetylcholine (ACh) release by adenosine is an important mechanism by which the secretory apparatus is regulated at both mammalian (Ginsborg and Hirst, 1972; Hirsh et al., 2002; Silinsky, 2004) and amphibian (Silinsky, 1980; Silinsky and Solsona, 1992; Redman and Silinsky, 1993, 1994; Robitaille et al., 1999) neuromuscular junctions (NMJs). ACh is known to be costored with ATP in cholinergic vesicles (Zimmermann, 1994), and it has been demonstrated that at amphibian NMJs, adenosine derived from neurally released ATPis the mediator of neuromuscular depression exhibited at low frequencies of nerve stimulation (Redman and Silinsky, 1994) (Fig. 1). At the mouse motor nerve ending the inhibitory actions of adenosine on transmitter release are linked to a reduction in the nerve-terminal calcium current associated with neurotransmitter release (Silinsky, 2004). In contrast, at the frog motor nerve, inhibition of ACh release by adenosine occurs in the absence of any effect on nerve-terminal calcium currents (Silinsky and Solsona, 1992; Redman and Silinsky, 1994; Robitaille et al., 1999). That is, at the frog NMJ adenosine inhibits ACh release through an effect on a process that takes place downstream from calcium entry. Although the exact site at which adenosine inhibits transmitter release is unknown, both the speed (50-100 ms; E. M. Silinsky, unpublished observations) and the stimulation-independent nature of inhibition suggest that this process must occur through an action on vesicles that are already primed and ready for release. Thus, the likely sites for mediating the action of adenosine are those core components of the neurotransmitter release process, the three SNARES (SNAP-25, syntaxin, and synaptobrevin), and synaptotagmin. However, there are difficulties in addressing which of these individual elements of the secretory apparatus might be involved in the actions of adenosine. We could use fractions of botulinum toxin to eliminate individual components of the secretory apparatus. However, each of these core components of the release machinery is individually essential for the neurotransmitter release process. Therefore, we decided to approach this problem by alternative means.

Modulation of Ca(2+)-dependent and Ca(2+)-independent miniature endplate potentials by phorbol ester and adenosine in frog.

Phorbol esters and adenosine modulate transmitter release from frog motor nerves through actions at separate sites downstream of calcium entry. However, it is not known whether these agents have calcium-independent sites of action. We therefore characterised calcium independent miniature endplate potentials (mepps) generated in response to 4-aminoquinaldine (4-AQ(A)) and then compared the modulation of these mepps by phorbol esters and adenosine with that of normal calcium dependent mepps. Application of 30 microM 4-AQ(A) resulted in the appearance of a population of mepps with amplitudes greater than twice the total population mode (mepp(>2M)). In the presence of 4-AQ(A), K(+) depolarisation or hypertonicity increased the numbers of normal amplitude mepps (mepp(N)) but had no effect on the frequency of mepp(>2M) events, suggesting that mepp(>2M) are not dependent on calcium. Treatment with the botulinum toxin (Botx) fractions C, D, or E (which selectively cleave syntaxin, synaptobrevin and SNAP-25, respectively) produced equivalent reductions in both normal and 4-AQ(A) induced mepps, suggesting that both mepp populations have equal dependence on the intact SNARE proteins. Phorbol dibutyrate (PDBu, 100 nM) increased the frequencies of both populations of mepps recorded in the presence of 4-AQ(A). Adenosine (25 microM) selectively reduced the numbers of mepp(N) with no effect on the frequency of mepp(>2M) events. These results suggest that mepp(>2M) events released in response to 4-AQ(A) are dependent on intact forms of syntaxin, synaptobrevin and SNAP-25, but unlike mepp(N) are independent of a functional calcium sensor. The selective action of adenosine, to reduce the numbers of normal amplitude mepps without effecting the frequency of mepp(>2M) events, suggests that adenosine normally inhibits transmitter release through a mechanism that is dependent on the presence of a functional calcium sensor.

Inhibition of spontaneous acetylcholine secretion by 2-chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction.

1. Previous studies have reported discrepancies in the potencies of A(1) adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A(1) receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2. CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 microM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 microM) produced highly statistically-significant reductions in spontaneous ACh release. 3. In the presence of the non-selective protein kinase inhibitor, H7 (50 microM), the potency of CADO was increased such that 1 microM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 microM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 microM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5. The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A(1) adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

Phorbol esters and neurotransmitter release: more than just protein kinase C?

This review focuses on the effects of phorbol esters and the role of phorbol ester receptors in the secretion of neurotransmitter substances. We begin with a brief background on the historical use of phorbol esters as tools to decipher the role of the enzyme protein kinase C in signal transduction cascades. Next, we illustrate the structural differences between active and inactive phorbol esters and the mechanism by which the binding of phorbol to its recognition sites (C1 domains) on a particular protein acts to translocate that protein to the membrane. We then discuss the evidence that the most important nerve terminal receptor for phorbol esters (and their endogenous counterpart diacylglycerol) is likely to be Munc13. Indeed, Munc13 and its invertebrate homologues are the main players in priming the secretory apparatus for its critical function in the exocytosis process.

Evidence for constitutively-active adenosine receptors at mammalian motor nerve endings.

A study was made to determine if constitutively active adenosine receptors are present at mouse motor nerve endings. In preparations blocked by low Ca(2+)/high Mg(2+) solution, 8-cyclopentyl-1,3,dipropylxanthine (CPX, 10-100 nM), which has been reported to be both an A(1) adenosine receptor antagonist and inverse agonist, produced a dose-dependent increase in the number of acetylcholine quanta released by a nerve impulse. Adenosine deaminase, which degrades ambient adenosine into its inactive congener, inosine, failed to alter the response to 100 nM CPX. 8-Cyclopentyltheophylline (CPT, 3 µM), a competitive inhibitor at A(1) adenosine receptors, prevented the increase in acetylcholine release produced by CPX. At normal levels of acetylcholine release, neither adenosine deaminase nor CPX affected acetylcholine release at low frequencies of nerve stimulation in (+)-tubocurarine blocked preparations. The results suggest that a proportion of the acetylcholine release process is controlled by constitutively active adenosine receptors at murine motor nerve endings, providing the first evidence for constitutive activity of G-protein-coupled receptors that modulate the function of mammalian nerve endings.

Mechanisms of neuromodulation as dissected using Sr2+ at motor nerve endings.

The use of binomial analysis as a tool for determining the sites of action of neuromodulators may be complicated by the nonuniformity of release probability. One of the potential sources for nonuniformity of release probability is the presence of multiple forms of synaptotagmins, the Ca2+ sensors responsible for triggering vesicular exocytosis. In this study we have used Sr2+, an ion whose actions may be restricted to a subpopulation of synaptotagmins, in an attempt to obtain meaningful estimates of the binomial parameters p (the probability of evoked acetylcholine [Ach] release) and n (the immediate available store of ACh quanta, whereby m = np). In contrast to results in Ca2+ solutions, binomial analysis of Sr2+-dependent release reveals a dramatically reduced dependence of n on extracellular Sr2+ concentrations. In Sr2+ solutions, blockade of potassium channels with 3,4-diaminopyridine increased m by an exclusive increase in p, whereas treatment with phorbol ester increased m solely by effects on n. The cyclic adenosine monophosphate (cAMP) analogue CPT-cAMP increased m by increasing both n and p. The effect of CPT-cAMP on p but not on n was blocked by protein kinase A (PKA) inhibitors, whereas the effect on n was mimicked by 8-CPT-2'-O-Me-cAMP, a selective agonist for exchange protein directly activated by cAMP, otherwise known as the cAMP-sensitive guanine nucleotide-exchange protein. The results demonstrate both the utility of the binomial distribution in Sr2+ solutions and the dual effects of cyclic AMP on both PKA-dependent and PKA-independent processes at the amphibian neuromuscular junction.

Modulation of calcium currents is eliminated after cleavage of a strategic component of the mammalian secretory apparatus.

Adenosine inhibits neurotransmitter secretion from motor nerves by an effect on the secretory apparatus in amphibia. In contrast, the inhibitory effect of adenosine is associated with decreases in calcium currents at mouse motor nerve endings. To determine if the action of adenosine in the mouse is mediated thorough a direct effect on calcium channels or through the secretory machinery, the effects of cleavage of the SNARE proteins on the action of adenosine were examined. Cleavage of the SNARE syntaxin with botulinum toxin type C (Botx/C) prevented the inhibitory effect of adenosine on nerve terminal calcium currents. Cleavage of the other SNAREs (synaptobrevin with Botx/D or SNAP-25 with Botx/A) failed to affect the inhibitory action of adenosine. The results provide evidence for an intimate coupling of nerve terminal calcium channels with a plasma membrane component of the SNARE complex, such that modulation of calcium currents by a G-protein coupled receptor cannot occur when syntaxin is cleaved.

Adenosine decreases both presynaptic calcium currents and neurotransmitter release at the mouse neuromuscular junction.

A controversy currently exists as to the mechanism of action by which adenosine, an endogenous mediator of neurotransmitter depression, reduces the evoked release of the neurotransmitter acetylcholine (ACh) at the skeletal neuromuscular junction. Specifically, it is uncertain whether adenosine inhibits ACh release from mammalian motor nerve endings by reducing Ca(2+) calcium entry through voltage-gated calcium channels or, as is the case at amphibian motor nerve endings, by an effect downstream of Ca(2+) entry. In an attempt to address this controversy, the effects of adenosine on membrane ionic currents and neurotransmitter release were studied at neuromuscular junctions in adult mouse phrenic nerve hemidiaphragm preparations. In wild-type mice, adenosine (500 microm-1 mm) reduced prejunctional Ca(2+) currents simultaneously with a reduction in evoked ACh release. In Rab3A knockout mice, which have been shown to have an increased sensitivity to adenosine, the simultaneous reduction in Ca(2+) currents and ACh secretion occurred at significantly lower adenosine concentrations (< or = 50 microM). Measurements of nerve terminal Na(+) and K(+) currents made simultaneously with evoked ACh release demonstrated that the decreases in Ca(2+) currents were not attributable to changes in cation entry through voltage-gated Na(+) or K(+) channels. Furthermore, no effects of adenosine on residual ionic currents were observed when P/Q-type calcium channels were blocked by Cd(2+) or omega-agatoxin-IVA. The results demonstrate that inhibition of evoked neurotransmitter secretion by adenosine is associated with a reduction in Ca(2+) calcium entry through voltage-gated P/Q Ca(2+) channels at the mouse neuromuscular junction. Whilst it may be that adenosine inhibits ACh release by different mechanisms at amphibia and mammalian neuromuscular junctions, it is also possible that the secretory apparatus is more intimately coupled to the Ca(2+) channels in the mouse such that an effect on the secretory machinery is reflected as changes in Ca(2+) currents.

Regulation by Rab3A of an endogenous modulator of neurotransmitter release at mouse motor nerve endings.

Rab3A, a small GTP-binding protein attached to synaptic vesicles, has been implicated in several stages in the process of neurosecretion, including a late stage occurring just prior to the actual release of neurotransmitter. The inhibitory neuromodulator adenosine also targets a late step in the neurosecretory pathway. We thus compared neuromuscular junctions from adult Rab3A(-/-) mutant mice with those from wild-type mice with respect to: (a) the basic electrophysiological correlates of neurotransmitter release at different stimulation frequencies, and (b) the actions of exogenous and endogenous adenosine on neurotransmitter release in normal calcium solutions. Neither the spontaneous quantal release of acetylcholine (ACh) nor basal evoked ACh release (0.05 Hz) differed between the mutant and wild-type mice. At 50-100 Hz stimulation (10-19 stimuli), facilitation of release was observed in the mutant mice but not in wild-type, followed by a depression of ACh release in both strains. ACh release at the end of the stimulus train in the mutant mouse was approximately double that of the wild-type mouse. The threshold concentration for inhibition of ACh release by exogenous adenosine was over 20-fold lower in the mutant mouse than in the wild-type mouse. The adenosine A(1) receptor antagonist 8-cyclopentyltheophylline (CPT) increased ACh release (0.05-1 Hz stimulation) in the mutant mouse under conditions in which it had no effect in the wild-type mouse. CPT had no effect on the pattern of responses recorded during repetitive stimulation in either strain. The results suggest that Rab3A reduces the potency of adenosine as an endogenous mediator of neuromuscular depression.