In the gastrointestinal tract, tachykinins are peptide neurotransmitters in nerve circuits that regulate intestinal motility, secretion, and vascular functions. Tachykinins also contribute to transmission from spinal afferents that innervate the gastrointestinal tract and have roles in the responses of the intestine to inflammation. Tachykinins coexist with acetylcholine, the primary transmitter of excitatory neurons innervating the muscle, and act as a co-neurotransmitter of excitatory neurons. Excitatory transmission is mediated through NK1 receptors (primarily on interstitial cells of Cajal) and NK2 receptors on the muscle. Tachykinins participate in slow excitatory transmission at neuro-neuronal synapses, through NK1 and NK3 receptors, in both ascending and descending pathways affecting motility. Activation of receptors (NK1 and NK2) on the epithelium causes fluid secretion. Tachykinin receptors on immune cells are activated during inflammation of the gut. Finally, tachykinins are released from the central terminals of gastrointestinal afferent neurons in the spinal cord, particularly in nociceptive pathways.

SUMMARY1. Single-channel studies were made using the patch-clamp technique of K channels in dispersed single smooth muscle cells from rabbit longitudinal jejunal muscle and guinea-pig small ( < 0-2 mm o.d.) mesenteric arteries.2. In isolated inside-out patches from these two types of smooth muscle cell there was a population of K channels which had single-channel conductances of about 100 pS in near physiological K gradients and about 200 pS with symmetrical 126 mM-K solutions. Their conductance and other properties distinguish them from a K channel of smaller conductance which we have previously described in these cells.3. The relative permeability of the channel with respect to K was 1-4 Tl: 1-0 K:0-7 Rb: < 0 05 Na: < 0 05 Cs. Cs (1 mm applied to the outside of the membrane) interfered with inward K movement when the membrane was hyperpolarized. Rb conductance of the channel when both sides of the membrane were exposed to 126 mm-Rb was 30 pS. Raising [Ca]i increased the mean duration of the (long) open state and decreased or had no effect on the duration of short, intermediate, and long mean closed states.

SUMMARY1. Membrane potential was recorded by micro-electrode in segments of small (200-500 jsm o.d.) mesenteric arteries of guinea-pig. Isotonic shortening was recorded in helical strips cut from these arteries.2. Raising the external potassium concentration, [K+]0, caused shortening and substantial depolarization. The threshold for contraction was about 30 mm which corresponded to a membrane potential of about -45 mV. Since high-potassium contractions were abolished in calcium-free solution it was suggested that they occur due to potential-sensitive calcium channels opening positive to about -45 mV.3. Noradrenaline weakly depolarized the muscle and produced contractions resistant to calcium-free conditions. It was suggested that noradrenaline contractions are mainly caused by mechanisms other than the opening of potential-sensitive calcium channels, namely entry of calcium via other channels and release of stored calcium.4. Carbachol had no effect on basal tension but inhibited shortening by noradrenaline or by raising [K+]0. The inhibitory effect of carbachol on tension under various conditions was associated with hyperpolarization or depolarization in a range negative to -45 mV, or no effect on potential, so that modulation of the number of open potential-sensitive calcium channels could not be evoked to explain its relaxant action.5. Removal or destruction of the endothelium by rubbing or by distilled water perfusion left tension responses to noradrenaline or raised [K+]. essentially unchanged. However, the inhibitory effect of carbachol on tension was attenuated and hyperpolarization of the resting artery was converted to a depolarization. 6. It was concluded that carbachol has both a strong inhibitory and a weak excitatory effect on these vascular smooth muscle cells. Membrane potential changes are not essential to its inhibitory action but may, by closing potential-sensitive calcium channels, sometimes reinforce it. Hyperpolarization by carbachol may be caused by a factor released by the action of carbachol on endothelial cells: in its absence carbachol may weakly depolarize but this alone is normally insufficient to generate tension.

SUMMARY1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea-pig ileum were used for recording membrane currents under whole-cell voltage clamp in response to carbachol (100 /bM, unless otherwise stated) or histamine (100 jam) applied extracellularly.2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca2+_ activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents.3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of -40 mV, which was considered to represent opening of cationic channels. The carbachol-induced inward current was much longer in duration and larger in size than the histamine-induced inward current.4. Inclusion of GDPpJS (2 mm) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol.5. When the holding potential was held at 0 mV with inclusion of GTPyS (0'1-1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break-through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTPyS had released Ca2+ stores. When the holding potential was held at -40 mV and GTPyS (0-1 or 0-2 mm) was present in the pipette, an inward current developed a few minutes after break-through. During the GTPyS-induced inward current, application of carbachol or histamine produced no further inward current. However, when 0-01 mmGTPyS was included in the pipette solution, carbachol-and histamine-induced inward currents were potentiated.6. Pretreated with 2-5 gug/ml pertussis toxin (PTX) did not change noticeably the MS 9646 106 S. KOMORI, M. KA WAI, T. TAKEWAKI AND H. OHASHI outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.7. The results suggest that stimulation of muscarinic receptor or histamine receptor caused release of Ca2+ from storage sites and activation of cationic channels, and that regardless of the receptor type, calcium store release may be mediated via a PTX-insensitive G-protein, while the cation channels are activated via another G-protein which is sensitive to PTX.

The interaction of Ba2+ and TEA with Ca2+-activated K+ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 micron external diameter). Ba2+ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba2+ into the channel and was reduced by increasing K+ ion concentration on the outside of the membrane. These results are consistent with Ba2+ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.

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1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied.2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP638, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 MM.3 PACAP638 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIPIO 28 at 3 Mm, which inhibited the relaxation by 44%.4 An antagonist of large conductance Ca2+-activated K+ channels, charybdotoxin, concentrationdependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM.5 An antagonist of small conductance Ca2 +-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 gM).6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP638 plus VIPI0O28. 7 The inhibitory effect of VIP1028 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM-1 Mm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation. VIPI0O28, but not PACAP638 selectively inhibited the VIP-induced relaxation.9 Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP638, but not VIPI0O28 selectively inhibited the PACAP-induced relaxation.10 Apamin at 100 nM inhibited inhibitory junction potentials (ij.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of ij.ps induced by two pulses at 10 Hz. VIPIO28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIPIO28 abolished the ij.ps induced by two pulses.11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin-and apaminsensitive K+ channels, respectively.