We examined the functional role of large-conductance Ca2+-activated K+(KCa) channels in the hamster cremasteric microcirculation by intravital videomicroscopy and characterized the single-channel properties of these channels in inside-out patches of membrane from enzymatically isolated cremasteric arteriolar muscle cells. In second-order (39 ± 1 μm, n = 8) and third-order (19 ± 2 μm, n = 8) cremasteric arterioles with substantial resting tone, superfusion with the KCa channel antagonists tetraethylammonium (TEA, 1 mM) or iberiotoxin (IBTX, 100 nM) had no significant effect on resting diameters ( P > 0.05). However, TEA potentiated O2-induced arteriolar constriction in vivo, and IBTX enhanced norepinephrine-induced contraction of cremasteric arteriolar muscle cells in vitro. Patch-clamp studies revealed unitary K+-selective and IBTX-sensitive currents with a single-channel conductance of 240 ± 2 pS between −60 and 60 mV ( n = 7 patches) in a symmetrical 140 mM K+ gradient. The free Ca2+ concentration ([Ca2+]) for half-maximal channel activation was 44 ± 3, 20 ± 1, 6 ± 0.4, and 3 ± 0.5 μM at membrane potentials of −60, −30, +30, and +60 mV, respectively ( n = 5), with a Hill coefficient of 1.9 ± 0.2. Channel activity increased e-fold for a 16 ± 1 mV ( n = 6) depolarization. The plot of log[Ca2+] vs. voltage for half-maximal activation ( V ½) was linear ( r 2 = 0.9843, n = 6); the change in V ½ for a 10-fold change in [Ca2+] was 84 ± 5 mV, and the [Ca2+] for half-maximal activation at 0 mV (Ca0; the Ca2+ set point) was 9 μM. Thus, in vivo, KCa channels are silent in cremasteric arterioles at rest but can be recruited during vasoconstriction. We propose that the high Ca0 is responsible for the apparent lack of activity of these channels in resting cremasteric arterioles, and we suggest that this may result from expression of unique KCa channels in the microcirculation.

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated from Ascaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also depended on external Ca++ and were reduced by the Ca(++)-channel blocker verapamil, observations consistent with the finding that nematode NOS is Ca(++)-dependent. KSAYMRFamide (PF3), KNIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A. suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

1. The physiological effects of two Phe-Met-Arg-Phe-NH2 (FMRFamide)-related neuropeptides isolated from the free-living nematode Panagrellus redivivus, SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2), were examined using neuromuscular preparations from the parasitic nematode Ascaris suum. 2. PF1 and PF2 hyperpolarized muscle membrane and induced sustained flaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. 3. PF1 reversed spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatory nematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. 4. PF1 reversal of levamisole contraction was blocked by pretreatment with agents that interfere with nitric oxide (NO) synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution, mimicked PF1 and PF2. 5. NO synthase activity, monitored by the conversion of [3H]arginine to [3H]citrulline, was twice as abundant in A. suum hypodermis as in muscle, but was not present in reproductive tissue. The relative abundance of NO synthase activity in these tissues was similar to the observed specific binding of [3H]PF1. 6. These results suggest that the inhibitory effects of PF1 and PF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role in this process analogous to that of the endothelium in vertebrate vasculature.

Two FM AFamide-related neuropeptides, GY-IRFamide and YlRFamide, were isolated from the marine turbellarian Bdelloura candida. The peptides elicited a dose-dependent contraction of isolated turbellarian muscle fibers, and both were more potent than FMRFamide. Structure-activity studies, using a range of analogues of the tetrapeptide amide, indicated that the structure of the endogenous peptides was optimal for peak activity. I mmunocytochem istry, using an autologous antiserum, revealed a widespread distribution of peptide immunoreactivity within central and peripheral neurons and their processes. This study indicates an important role for GY-IRFamide and YlRFamide in the control of neuromuscular function in turbellarians.