Translation of the rat thoracic contusion model; part 2 - forward versus backward locomotion testing.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Locomotion analyses in rat spinal cord contusion injury (SCI) models are widely used for the evaluation of recovery of supraspinal locomotor control. However, many commonly used locomotion tests are inadequate to test for spinal cord integrity as they assess motor function that can be highly mediated through below-level propriospinal pattern-generating circuitry, independently of below-level perception. Here we report a behavioral motor test that is more sensitive for spinal cord integrity, even 6 weeks after injury: the backward locomotion rotating rod. SETTING: University of California - San Diego. METHODS: A modified rotating rod test was run in reverse. The rod diameter was increased and thin rubber lining was added. As a reference, we included commonly used motor tests: BBB score, catwalk gait analysis, motor-evoked potentials, single frame analyses, a forward rotating rod test and the 55° inclined ladder test. RESULTS: Unlike commonly used motor tests, the backward locomotion rotating rod test significantly discriminates between both sham-operated (falling latency: 20.4 s s.d.±4.5) vs mild SCI animals, and mild vs moderate SCI animals (differences between each group at acute, subacute and chronic phases: ⩾6 s, P⩽0.01). Moderate SCI animals were practically unable to make even slight backward hindpaw movements. The backward locomotion ability in the chronic phase correlates best with BBB locomotor scores from the acute phase. CONCLUSION: Our data show that backward locomotion is a highly sensitive and quick test to discriminate between sham, mild and moderate SCI, even after 6 weeks. Backward locomotion testing may improve the translational value of experimental results for the clinic.

Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Potent suppression of stretch reflex activity after systemic or spinal delivery of tizanidine in rats with spinal ischemia-induced chronic spastic paraplegia.

BACKGROUND: Spasticity and rigidity are serious complications associated with spinal traumatic or ischemic injury. Clinical studies show that tizanidine (Tiz) is an effective antispasticity agent; however, the mechanism of this effect is still not clear. Tiz binds not only to α2-adrenoreceptors (AR) but also to imidazoline (I) receptors. Both receptor systems (AR+I) are present in the spinal cord interneurons and α-motoneurons. The aim of the present study was to evaluate the therapeutic potency of systematically or spinally (intrathecally [IT]) delivered Tiz on stretch reflex activity (SRA) in animals with ischemic spasticity, and to delineate supraspinal or spinal sites of Tiz action. EXPERIMENTAL PROCEDURES: Animals were exposed to 10 min of spinal ischemia to induce an increase in SRA. Increase in SRA was identified by simultaneous increase in recorded electromyography (EMG) activity and ankle resistance measured during computer-controlled ankle dorsiflexion (40°/3 s) in fully awake animals. Animals with increased SRA were divided into several experimental subgroups and treated as follows: (i) Tiz administered systemically at the dose of 1 mg kg(-1), or IT at 10 µg or 50 µg delivered as a single dose; (ii) treatment with systemic Tiz was followed by the systemic injection of vehicle, or by nonselective AR antagonist without affinity for I receptors; yohimbine (Yoh), α2A AR antagonist; BRL44408 (BRL), α2B AR antagonist; ARC239 (ARC), nonselective AR and I(1) receptor antagonist; efaroxan (Efa), or nonselective AR and I(2) receptor antagonist; idazoxan (Ida); (iii) treatment with IT Tiz was followed by the IT injection of selective α2A AR antagonist; atipamezole (Ati). In a separate group of spastic animals the effect of systemic Tiz treatment (1 mg/kg) or isoflurane anesthesia on H-reflex activity was also studied. RESULTS: Systemic and/or IT treatment with Tiz significantly suppressed SRA. This Tiz-mediated anti-SRA effect was reversed by BRL (5 mg kg(-1)), Efa (1 mg kg(-1)), and Ida (1 mg kg(-1)). No reversal was seen after Yoh (3 mg kg(-1)) or ARC (5 mg kg(-1)) treatment. Anti-SRA induced by IT Tiz (50 µg) was reversed by IT injection of Ati (50 µg). Significant suppression of H-reflex was measured after systemic Tiz treatment (1 mg/kg) or isoflurane (2%) anesthesia, respectively. Immunofluorescence staining of spinal cord sections taken from animals with spasticity showed upregulation of α2A receptor in activated astrocytes. CONCLUSIONS: These data suggest that α2A AR and I receptors, but not α2B AR, primarily mediate the Tiz-induced antispasticity effect. This effect involves spinal and potentially supraspinal sites and likely targets α2A receptor present on spinal neurons, primary afferents, and activated astrocytes. Further studies using highly selective antagonists are needed to elucidate the involvement of specific subtypes of the AR and I receptors in the antispasticity effect seen after Tiz treatment.

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.

c-Jun decreases voltage-gated K(+) channel activity in pulmonary artery smooth muscle cells.

BACKGROUND: Activity of voltage-gated K(+) (K(v)) channels controls membrane potential (E(m)) that regulates cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) by regulating voltage-dependent Ca(2+) channel function. A rise in [Ca(2+)](cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers vasoconstriction and stimulates PASMC proliferation. Whether c-Jun, a transcription factor that stimulates cell proliferation, affects K(v) channel activity in PASMCs was investigated. METHODS AND RESULTS: Infection of primary cultured PASMCs with an adenoviral vector expressing c-jun increased the protein level of c-Jun and reduced K(v) currents (I(K(V))) compared with control cells (infected with an empty adenovirus). Using single-cell reverse transcription-polymerase chain reaction, we observed that the mRNA level of Kv1.5 and the current density of I(K(V)) were both attenuated in c-jun-infected PASMCs compared with control cells and cells infected with antisense c-jun. Overexpression of c-Jun also upregulated protein expression of Kvbeta(2) and accelerated I(K(V)) inactivation. Furthermore, E(m) was more depolarized and [(3)H]thymidine incorporation was greater in PASMCs infected with c-jun than in control cells and cells infected with antisense c-jun. CONCLUSIONS: These results suggest that c-Jun-mediated PASMC proliferation is associated with a decrease in I(K(V)). The resultant membrane depolarization increases [Ca(2+)](cyt) and enhances PASMC growth.

Augmented K(+) currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis.

The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K(+) (K(V)) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented K(V) current [I(K(V))], and induced mitochondrial membrane depolarization. High K(+) (40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced I(K(V)) increase and apoptosis. Blockade of K(V) channels with 4-aminopyridine diminished I(K(V)) and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in I(K(V)). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal K(V) channels and mitochondrial membrane depolarization. The increased I(K(V)) would result in an apoptotic volume decrease due to a loss of cytosolic K(+) and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of K(V) channels would thus attenuate PASMC apoptosis.

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells.

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.

Inhibition of K(+) channel activity in human pulmonary artery smooth muscle cells by serum from patients with pulmonary hypertension secondary to congenital heart disease.

Activity of K(+) channels regulates cytosolic free Ca(2+) concentration by controlling membrane potential. A rise in cytosolic free Ca(2+) concentration in pulmonary artery smooth muscle cells (PASMC) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Whether serum from children with pulmonary hypertension (PH) secondary to congenital cardiopulmonary diseases contains a factor(s) that inhibits K(+) channel function in PASMC was investigated using patch clamp techniques. PASMC isolated from normal subjects were cultured in media containing 5% serum from normotensive (NPH) or PH patients. Cell growth rate and the currents through voltage-gated K(+) channels were determined and compared between the cells treated with serum from NPH and PH patients. In the absence of growth factors, incubation of PASMC in media containing NPH serum for 48 h increased cell numbers by 2.5-fold, whereas incubation of the cells in media containing PH serum increased cell numbers by 4.5-fold (p < 0.001). Amplitude of whole-cell voltage-gated K(+) currents in NPH serum-treated cells (1119 +/- 222 pA at +80 mV, n = 43) was 3.5-fold greater than in PH serum-treated cells (323 +/- 34 pA, n = 43, p < 0.001). Consistently, membrane potential was much more depolarized in PASMC treated with PH serum (-28 +/- 2 mV, n = 29) than cells treated with NPH-serum (-47 +/- 2 mV, n = 28; p < 0.001). These results suggest that a circulating mitogenic agonist, which induces membrane depolarization by inhibiting voltage-gated K(+) channel activity in PASMC, may be produced or up-regulated in pediatric patients with secondary PH.

Capacitative Ca(2+) entry in agonist-induced pulmonary vasoconstriction.

Agonist-induced increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca(2+) release from intracellular stores followed by a sustained Ca(2+) influx. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), which contributes to the sustained increase in [Ca(2+)](cyt) and the refilling of Ca(2+) into the stores. In isolated PAs superfused with Ca(2+)-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca(2+)](cyt) due to Ca(2+) release from the intracellular stores. The transient contraction lasted for 3-4 min until the Ca(2+) store was depleted. Restoration of extracellular Ca(2+) in the presence of phentolamine produced a contraction potentially due to a rise in [Ca(2+)](cyt) via CCE. The store-operated Ca(2+) channel blocker Ni(2+) reduced the store depletion-activated Ca(2+) currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca(2+) channels, plays an important role in agonist-mediated PA contraction.

Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes.

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.

Activation of K+ channels induces apoptosis in vascular smooth muscle cells.

Intracellular K+ plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and inhibiting apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+ concentration is mainly regulated by K+ uptake via Na+-K+-ATPase and K+ efflux through K+ channels in the plasma membrane. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H+ gradient across the inner membrane of mitochondria, induces apoptosis in many cell types. In rat and human pulmonary artery smooth muscle cells (PASMC), FCCP opened the large-conductance, voltage- and Ca2+-sensitive KK+ (maxi-K) channels, increased K+ currents through maxi-K channels [I(K(Ca))], and induced apoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM) decreased I(K(Ca)) by blocking the sarcolemmal maxi-K channels and inhibited the FCCP-induced apoptosis in PASMC cultured in media containing serum and growth factors. Furthermore, inhibition of K+ efflux by raising extracellular K+ concentration from 5 to 40 mM also attenuated PASMC apoptosis induced by FCCP and the K+ ionophore valinomycin. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to an increase of maxi-K channel activity. The resultant K+ loss through opened maxi-K channels may serve as a trigger for cell shrinkage and caspase activation, which are major characteristics of apoptosis in pulmonary vascular smooth muscle cells.

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration.

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation.

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation.

Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E(m)) regulates [Ca(2+)](cyt) by governing Ca(2+) influx through voltage-dependent Ca(2+) channels. Thus intracellular Ca(2+) may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K(+) (Kv) channels regulates resting E(m). In this study, we investigated whether changes of Kv currents [I(K(V))], E(m), and [Ca(2+)](cyt) affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca(2+) abolished PASMC growth. Resting [Ca(2+)](cyt) was significantly higher, and resting E(m) was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell I(K(V)) was significantly attenuated in PASMC during proliferation. Furthermore, E(m) depolarization significantly increased resting [Ca(2+)](cyt) and augmented agonist-mediated rises in [Ca(2+)](cyt) in the absence of extracellular Ca(2+). These results demonstrate that reduced I(K(V)), depolarized E(m), and elevated [Ca(2+)](cyt) may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca(2+)](cyt) in PASMC.

Effect of inorganic and organic tin compounds on ACh- and voltage-activated Na currents.

1. Inorganic tin and organotin compounds, occurring in aquatic ecosystems, are toxic and can cause behavioral abnormalities in living organisms. To determine the possible neuronal basis of these actions, the effects of both forms of Sn were studied on identified neurones of the mollusk, Lymnaea stagnalis L. 2. SnCl2 caused a dose-dependent decrease in the acetylcholine (Ach)-induced inward current. The effective threshold concentration, measured by a two microelectrode voltage clamp technique, was 0.1 microM, and the maximal effect occurred at 5 microM SnCl2. The depression of the inward current was greater after a 10 min preapplication (20%) than after 3 min treatment (7%). 3. The next series of experiments compared the actions of inorganic or organic tin compounds. In whole cell clamp experiments both (CH3)2SnCl2 and (CH3)3SnCl, like inorganic Sn, decreased the amplitude of Ach-induced current. Increasing the duration of the preapplication time resulted in an increase in the effect, but the action was not reversible. SnCl2 treatment caused a concentration-dependent alteration (initial potentiation followed by depression) of the amplitude of I(Na(V)) over the whole voltage range and slightly shifted the I-V curves to the left. In contrast, trimethyl tin decreased the amplitude of I(Na(V)) only at high concentration (100 microM). The activation time course of I(Na) was increased (tau = 0.43 ms in control and 0.55 ms in Sn), but Sn did not alter the inactivation parameters (tau = 3.43 and 3.41 ms). 4. These results support earlier findings that agonist- and voltage-activated channels are direct targets of toxic metals. We conclude that tin in both inorganic and organic forms acts at neuronal membranes to modulate synaptic transmission through direct actions on agonist-activated ion channels, and suggest that these actions may be the basis of the altered behavior of animals in tin-polluted environments.

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration.

Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K(+) channels (Kv) controls membrane potential (E(m)) that regulates cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) by governing the driving force for Ca(2+) influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K(+) channel gene expression, E(m), and [Ca(2+)](cyt) in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, alpha-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca(2+) channels, the depolarized E(m) in DFMO-treated cells decreased [Ca(2+)](cyt) as a result of reduced driving force for Ca(2+) influx through capacitative Ca(2+) entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E(m), and [Ca(2+)](cyt) but also restored cell migration to normal. Removal of extracellular Ca(2+) or blockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca(2+)](cyt) by increasing the driving force (the electrochemical gradient) for Ca(2+) influx and thus stimulates cell migration.

High K(+)-induced membrane depolarization attenuates endothelium-dependent pulmonary vasodilation.

Impairment of endothelium-dependent pulmonary vasodilation has been implicated in the development of pulmonary hypertension. Pulmonary vascular smooth muscle cells and endothelial cells communicate electrically through gap junctions; thus, membrane depolarization in smooth muscle cells would depolarize endothelial cells. In this study, we examined the effect of prolonged membrane depolarization induced by high K(+) on the endothelium-dependent pulmonary vasodilation. Isometric tension was measured in isolated pulmonary arteries (PA) from Sprague-Dawley rats, and membrane potential was measured in single PA smooth muscle cells. Increase in extracellular K(+) concentration from 4.7 to 25 mM significantly depolarized PA smooth muscle cells. The 25 mM K(+)-mediated depolarization was characterized by an initial transient depolarization (5-15 s) followed by a sustained depolarization that could last for up to 3 h. In endothelium-intact PA rings, ACh (2 microM), levcromakalim (10 microM), and nitroprusside (10 microM) reversibly inhibited the 25 mM K(+)-mediated contraction. Functional removal of endothelium abolished the ACh-mediated relaxation but had no effect on the levcromakalim- or the nitroprusside-mediated pulmonary vasodilation. Prolonged ( approximately 3 h) membrane depolarization by 25 mM K(+) significantly inhibited the ACh-mediated PA relaxation (-55 +/- 4 vs. -29 +/- 2%, P < 0.001), negligibly affected the levcromakalim-mediated pulmonary vasodilation (-92 +/- 4 vs. -95 +/- 5%), and slightly but significantly increased the nitroprusside-mediated PA relaxation (-80 +/- 2 vs. 90 +/- 3%, P < 0. 05). These data indicate that membrane depolarization by prolonged exposure to high K(+) concentration selectively inhibited endothelium-dependent pulmonary vasodilation, suggesting that membrane depolarization plays a role in the impairment of pulmonary endothelial function in pulmonary hypertension.