Pre- and postsynaptic contributions of voltage-dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons.

Activation of voltage-dependent Ca2+ channels (VDCCs) is critical for neurotransmitter release, neuronal excitability and postsynaptic Ca2+ signalling. Antagonists of VDCCs can be antinociceptive in different animal pain models. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the processing of pain-related information, but the role of VDCCs to the activity-dependent Ca2+ increase in lamina I neurons and to the synaptic transmission between nociceptive afferents and second order neurons in lamina I is not known. This has now been investigated in a lumbar spinal cord slice preparation from young Sprague-Dawley rats. Microfluorometric Ca2+ measurements with fura-2 have been used to analyse the Ca2+ increase in lamina I neurons after depolarization of the cells, resulting in a distinct and transient increase of the cytosolic Ca2+ concentration. This Ca2+ peak was reduced by the T-type channel blocker, Ni2+, by the L-type channel blockers, nifedipine and verapamil, and by the N-type channel blocker, omega-conotoxin GVIA. The P/Q-type channel antagonist, omega-agatoxin TK, had no effect on postsynaptic [Ca2+]i. The NMDA receptor channel blocker D-AP5 reduced the Ca2+ peak, whereas the AMPA receptor channel blocker CNQX had no effect. Postsynaptic currents, monosynaptically evoked by electrical stimulation of the attached dorsal roots with C-fibre and Adelta-fibre intensity, respectively, were reduced by N-type channel blocker omega-conotoxin GVIA and to a much lesser extent, by P/Q-type channel antagonist omega-agatoxin TK, and the L-type channel blockers verapamil, respectively. No difference was found between unidentified neurons and neurons projecting to the periaqueductal grey matter. This is the first quantitative description of the relative contribution of voltage-dependent Ca2+ channels to the synaptic transmission in lamina I of the spinal dorsal horn, which is essential in the processing of pain-related information in the central nervous system.

Reduction of glycine receptor-mediated miniature inhibitory postsynaptic currents in rat spinal lamina I neurons after peripheral inflammation.

Peripheral inflammation may induce long-lasting sensitization in the central nociceptive system. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the integration and relay of pain-related information. In rats we studied whether changes in passive and active membrane properties and/or alteration of glycine receptor-mediated inhibitory control of spinal lamina I neurons may contribute to central sensitization in a model of peripheral long-lasting inflammation (complete Freund's adjuvant, hindpaw). Spontaneously occurring glycine receptor-mediated miniature inhibitory postsynaptic currents (GlyR-mediated mIPSCs) were recorded in lumbar spinal lamina I neurons. Miniature IPSC rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected by peripheral inflammation. The mean frequency of GlyR-mediated mIPSCs of lamina I neurons ipsilateral to the inflamed hindpaw was, however, significantly reduced by peripheral inflammation when compared with neurons from noninflamed animals. Principal passive and active membrane properties and firing patterns of spinal lamina I neurons were not changed by inflammation. These results indicate that long-lasting peripheral inflammation leads to a reduced glycinergic inhibitory control of spinal lamina I neurons by a presynaptic mechanism.

Cell volume-induced changes in K+ transport across the rat colon.

The effect of cell swelling and cell shrinkage on K+ transport across the rat colonic epithelium was studied by measuring unidirectional fluxes, uptake and efflux of 86Rb+, a marker for K+. Exposure to a hypotonic medium stimulated the secretory, serosa-to-mucosa flux of K+, whereas exposure to a hypertonic medium inhibited the absorptive, mucosa-to-serosa flux of K+ in the distal, but not in the proximal colon. Neither manoeuvre had any effect on the uptake of K+ across the apical or the basolateral membrane. Cell swelling induced a sustained increase in the apical and basolateral K+ efflux from both colonic segments, whereas cell shrinkage reduced the efflux. Ba2+ (10(-2) mol l(-1)) inhibited the swelling-induced stimulation of the apical, quinine (10(-3) mol l(-1)) that of the basolateral K+ efflux in the distal colon. Incubation of the tissue in Ca2+-free buffer or La3+, which blocks Ca2+-influx into the epithelium, strongly reduced the basal K+ efflux across the basolateral membrane. The same was observed with brefeldin A, a blocker of the transport of newly synthesized proteins out of the endoplasmatic reticulum. Swelling-induced K+ efflux, however, was not reduced. In the presence of colchicine, an inhibitor of the polymerization of microtubules, swelling evoked only a transient increase in mucosal efflux, which, especially in the proximal colon, fell after 6 min to the level of the isotonic control period. These results demonstrate that the cell volume is involved in the regulation of transepithelial K+ transport across the rat colonic epithelium and suggest a role of the cytoskeleton in the control of a part of the volume-sensitive K+ channels.

Spontaneous contractions of intestinal smooth muscle re-aggregates from the new-born rat triggered by thromboxane A2.

Isolated smooth muscle cells from the small intestine of new-born rats were prepared by enzymatic digestion. These cells re-aggregate after 1 day in culture to clusters. The re-aggregates show spontaneous rhythmical contractions at 37 degrees C with a frequency (13.1 +/- 0.8 min-1, n = 49), which is similar to that of the intact smooth muscle layer. The cholinergic agonist carbachol (5 x 10(-5) mol l-1) caused an increase in the frequency of the spontaneous contractions often ending in a permanent contraction. A similar effect was achieved with the thromboxane A2 (TXA2) agonist, U-46619 (10(-5) mol l-1). In contrast, both the TXA2 receptor blocker, Bay u3405 (5 x 10(-4) mol l-1), as well as the Ca2+ channel blocker, verapamil (5 x 10(-5) mol l-1), suppressed the spontaneous contractions. The observed contractility was insensitive against the neuronal blocker tetrodotoxin (10(-6) mol l-1). These analyses of video images were supported by the measurement of relative changes in the intracellular Ca2+ concentration with the Ca(2+)-sensitive dye, fura-2. Spontaneous contractions were paralleled by spikes in the intracellular Ca2+ concentration, which were abolished by Bay u3405, but stimulated by U-46619 or carbachol. In summary, these results obtained at re-aggregates of intestinal smooth muscle cells support the hypothesis of a role of TXA2 in the generation of spontaneous intestinal smooth muscle contractions in vitro.

Activation of group I metabotropic glutamate receptors induces long-term depression at sensory synapses in superficial spinal dorsal horn.

Low-frequency stimulation of primary afferent Adelta-fibers can induce long-term depression of synaptic transmission in rat superficial spinal dorsal horn. Here, we have identified another form of long-term depression in superficial spinal dorsal horn neurons that is induced by specific group I but not group II metabotropic glutamate receptor (mGluR) agonists. Synaptic strength between Adelta-fibers and dorsal horn neurons was examined by intracellular recordings in a spinal cord-dorsal root slice preparation from young rat. In the presence of bicuculline and strychnine, bath application of (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid ((1S,3R)-ACPD) or the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) but not the specific group II mGluR agonist (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV) for 20 min produced an acute and a long-term depression of synaptic strength. Bath application of the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovaleric acid did not affect these depressions by (1S,3R)-ACPD. After pre-incubation of slices with pertussis toxin, a G-protein inhibitor, (1S,3R)-ACPD still induced acute and long-term depressions. The phospholipase C inhibitor U73122 stereoselectively blocked the induction of long-term depression without affecting acute synaptic inhibition. This study demonstrates that, in the spinal cord, direct activation of group I mGluRs that are coupled to phospholipase C through pertussis toxin-insensitive G-proteins induces a long-term depression of synaptic strength. This may be relevant to the processing of sensory information in the spinal cord, including nociception.

Involvement of calmodulin and protein kinase C in the regulation of K+ transport by carbachol across the rat distal colon.

The cholinergic agonist carbachol stimulates the apical H+-K+-ATPase and apical as well as basolateral K+ channels in the rat distal colon. The effect of carbachol was tested in the presence of different inhibitors of the Ca2+ signaling pathway in order to characterize the intracellular mechanisms involved. Both carbachol-stimulated Rb+-efflux as well as carbachol-stimulated mucosal Rb+-uptake were dependent on the presence of serosal Ca2+. The Ca2+-calmodulin antagonist calmidazolium (10(-7) mol l(-1)) inhibited the stimulation of mucosal and serosal Rb+ efflux by carbachol. A similar effect had KN-62 (10(-5) mol l(-1)), an inhibitor of the Ca2+-calmodulin-dependent kinase II, suggesting the regulation of basolateral and apical K+ channels by this kinase. Staurosporine (10(-6) mol l(-1)), which potently inhibits protein kinase C, did not alter the effect of carbachol on Rb+ efflux, although the stimulation of apical Rb+ efflux by carbachol seemed to be less prolonged, indicating that protein kinase C is not involved in the regulation of K+ permeability. In contrast, mucosal Rb+ uptake, which is determined by the ouabain- and vanadate-sensitive K+ transport via the apical H+-K+-ATPase, was decreased to nearly one third of control values in the presence of calmidazolium. Both calmidazolium and staurosporine, but not KN-62, prevented the stimulatory action of carbachol on the H+-K+-ATPase, suggesting a synergistic control of this ion pump by both Ca2+-calmodulin and protein kinase C.

The protein tyrosine kinase pathway is not involved in the regulation of K+ transport across the rat colon.

The protein tyrosine kinase inhibitor, genistein, is known to activate the cystic fibrosis transmembrane regulator (CFTR) Cl- channel and to inhibit K+ currents across the rat colonic epithelium. The aim of the present study is to answer the question whether these effects are involved in the regulation of transepithelial K+ transport. Therefore, the action of genistein on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake and efflux of Rb+ in mucosa-submucosa preparations. All effects of genistein (5 x 10(-5) mol L(-1)) were tested in the presence of a low concentration of forskolin (2 x 10(-7) mol L(-1)), because prestimulation of the cAMP pathway has been shown to be a prerequisite for a secretory action of genistein. Forskolin caused an increase in the serosa-to-mucosa flux of Rb+ (J(Rb)sm) thereby stimulating net K+ secretion in the proximal and distal colon. None of these effects was further enhanced after administration of genistein. Neither mucosal uptake of Rb+, representing mainly the activity of the H+-K+-ATPase in the distal colon, nor serosal Rb+ uptake, representing, e.g. the activity of the Na+-K+-2Cl- cotransporter, were affected by genistein. Also the efflux of Rb+ across the apical or the basolateral membrane, an indicator for the apical and basolateral K+ conductance, was unchanged in the presence of genistein. These results demonstrate that the K+ channels inhibited by genistein are not involved in transepithelial K+ transport.

Potassium conductances in isolated single cells from Xenopus laevis colonic epithelium.

The patch-clamp technique was employed in whole cells to analyze K+ conductances of amphibian colonic cells. Xenopus laevis colonic epithelium was dissected, and single epithelial cells were isolated using Ca(2+)-free solution and mild enzyme treatment. Vital epithelial cells had a round shape, and a distinction between apical and basolateral poles was no longer possible. Their epithelial origin was, however, verified by antibodies against keratin. The average resting potential of the colonocytes was -37.6 +/- 1 mV (n = 220) and the resulting membrane current was strongly potassium selective. Further characterization of this conductance was achieved by current-voltage relationship in the presence and absence of various K+ channel blockers. Barium and cesium showed pronounced voltage-dependent blockage, with interaction at about 35% inside the pores. Lidocain, as well as quinine and quinidine also blocked, but with different kinetics and binding charactertics. Both TEA and verapamil were ineffective. We also explored the effects of extra- (pHo) and intracellular pH (pHi) on the K+ conductance. An increase of pHo, as well as pHi, caused membrane hyperpolarization, and the shift of the current-voltage relationship indicates a stimulation of K+ channels by decreasing external and/or internal H+ concentration. The results provide the first whole-cell measurements on isolated amphibian colonic epithelial cells and demonstrate the presence of various K+ channel types in this preparation.

Mechanisms of carbachol-induced alterations in K+ transport across the rat colon.

The effect of carbachol, an agonist of the Ca2+ pathway, on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake, and efflux of Rb+, a marker for K+, in mucosa-submucosa preparations. Unidirectional ion flux measurements revealed that carbachol stimulated K+ secretion in the proximal colon by a marked increase in the serosa-to-mucosa flux (J(Rb)sm) and a more moderate rise in the mucosa-to-serosa flux (J(Rb)ms). In the distal colon carbachol had no effect on J(Rb)ms but J(Rb)sm was reduced after a transient increase finally resulting in an inhibition of K+ secretion. Carbachol caused a stimulation of mucosal Rb+ uptake in the distal colon, which was diminished in the presence of inhibitors of the apical H+-K+-ATPase, vanadate and ouabain. In contrast, in the proximal colon the serosal Rb+ uptake was enhanced by carbachol, an effect, which could be prevented by bumetanide, an inhibitor of the basolateral Na+-K+-2Cl(-)-cotransporter. Efflux experiments revealed that carbachol caused a transient increase of apical and basolateral Rb+ permeability in both colonic segments. In the distal colon, stimulated K+ efflux to the serosal side was reduced by quinine, efflux to the mucosal side was blocked by tetraethylammonium. In the proximal colon, carbachol-activated apical and basolateral K+ efflux were inhibited by Ba2+. In conclusion, these data suggest that in the distal colon carbachol stimulates the H+-K+-ATPase and the basolateral K+ efflux through quinine-sensitive K+ channels, whereas in the proximal colon carbachol induces K+ secretion due to a stimulation of the basolateral Na+-K+-2Cl(-)-cotransporter and an increased efflux to the luminal side via Ba2+-sensitive apical K+ channels.

In vitro analysis of synergistic effects of fibrinolytic agents and prostacyclin analogues.

We investigated the in vitro thrombolytic effects of streptokinase, urokinase, alteplase and saruplase, alone or in combination, with the prostacyclin analogues, iloprost and taprostene. Human platelet-rich plasma was stimulated with collagen (1 microgram/ml) to generate thrombi containing platelets and fibrin. Following treatment with fibrinolytic agents, lysis was allowed to proceed for 30 min and was then terminated with aprotinin (2,000 CIU/ml). To evaluate the combinatory effects of fibrinolytic agents and prostacyclin analogues, we used concentrations of fibrinolytic agents which reduced thrombi weight by less than 50%. Neither iloprost nor taprostene alone demonstrated any thrombolytic effects. Furthermore, the thrombolytic efficacies of streptokinase, alteplase and saruplase were not enhanced by prostacyclin analogues. The thrombolytic activities of alteplase and urokinase, however, were additive. Similarly, the combination of urokinase with saruplase had an additive thrombolytic effect in our in vitro model. The effects of alteplase and saruplase were synergistic at a molar ratio of 1:4.7.

Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus.

We present a detailed analysis of the content and organization of the human immunoglobulin VH locus. Human VH genes representing five distinct families were isolated, including novel members belonging to two out of three of the known VH gene families (VH1 and VH3) as well as members of three new families (VH4, VH5, and VH6). We report the nucleotide sequence of 21 novel human VH genes, many of which belong to the three new VH gene families. In addition, we provide a preliminary analysis of the organization of these gene segments over the full extent of the locus. We find that the five multi-segment families (VH1-5) have members interspersed over nearly the full 1500-2000 kb of the VH locus, and estimate that the entire heavy chain locus covers 2500 kb or less. Finally, we provide the first report of the physical linkage of the variable and constant loci of a human Ig gene family by demonstrating that the most proximal known human VH segments lie within 100 kb of the constant region locus.