Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis.

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.

Suppression of the rat microglia Kv1.3 current by src-family tyrosine kinases and oxygen/glucose deprivation.

Microglia activate following numerous acute insults to the brain, including oxygen/glucose deprivation (OGD), and both protein tyrosine kinases (PTKs) and K+ channels have been implicated in their activation. We identified Kv1.3 (voltage-gated potassium channel) protein in cultured rat microglia and confirmed that the native current is biophysically and pharmacologically similar to Kv1. 3. To explore whether src-family PTKs regulate the microglial Kv current, we first heterologously expressed Kv1.3 in a microglia-like cell line derived from neonatal rat brain (MLS-9). The resulting large Kv1.3 current was eliminated by co-transfecting the constitutively active PTK, v-src, then rapidly restored by the PTK inhibitor, lavendustin A. Acute activation of endogenous src kinases by a peptide activator significantly reduced the current, an effect that was mimicked by OGD. Similarly, in primary cultures of rat microglia, the endogenous Kv1.3-like current was inhibited by activating endogenous src-family PTKs and by OGD. Biochemical analysis showed that OGD increased the tyrosine phosphorylation of native Kv1.3 protein, which was alleviated by PTK inhibitors or reactive oxygen species (ROS) scavengers. Conversely, the basal level of Kv1.3 phosphorylation was decreased by PTK inhibitors or scavengers of ROS. Together, our results point to a post-insertional downregulation of the microglial Kv1.3-like current by oxidative stress and tyrosine phosphorylation. This interaction may be facilitated by a multiprotein complex because, in cultured microglia, the endogenous Kv1.3 and src proteins both bind to the scaffolding protein, post-synaptic density protein 95 (PSD-95). By associating with, and phosphorylating Kv1.3, src is well positioned to regulate microglial responses to oxidative stress.

Role of gap junctions in structural arrangements of interstitial cells of Cajal and canine ileal smooth muscle.

We examined the structural and functional basis for pacemaking by interstitial cells of Cajal (ICC) in circular smooth muscle of the canine ileum. Gap junctions were found between ICC of myenteric plexus (MyP), occasionally between MyP ICC and outer circular smooth muscle cells, between individual outer circular smooth muscle cells, between them and ICC of the deep muscular plexus (DMP), and between DMP ICC. No visible gap junctions connected MyP ICC to longitudinal muscle cells or inner circular muscle cells. Occasionally contacts occurred between the two muscle layers. No special structures were found to connect MyP and DMP ICC networks. Octanol concentration dependently reduced the amplitude and frequency of, but did not abolish, slow waves in circular muscle in isolated ileum recorded near the MyP or the DMP. Slow waves triggered from MyP ICC by a current pulse also persisted. Contractile activity was abolished, cells were depolarized, and fast inhibitory junction potentials were reduced by octanol. We conclude that ICC pacemakers of the MyP and DMP utilize gap junctional conductances for pacemaking function but may not require them. Coupling between the two ICC networks may utilize the circular muscle syncytium.

HERG-like K+ channels in microglia.

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

Influence of nitric oxide and vasoactive intestinal peptide on the spontaneous and triggered electrical and mechanical activities of the canine ileum.

Modulation of canine ileal pacemaker activity by nitric oxide (NO) or vasoactive intestinal peptide (VIP) was studied during recording of the intracellular electrical and mechanical activity from the entire muscularis externa and from an isolated circular muscle preparation both cut in the long axis of the circular muscle. In the whole-thickness preparation with cholinergic and adrenergic nerve function blocked, the inhibitory junction potentials (IJPs) recorded near the myenteric plexus (MyP) or deep muscular plexus (DMP) were abolished by omega-conotoxin GVIA (omega-CTX, 10(-7) to 3 x 10(-7) M), tetrodotoxin (TTX, 1 microM), or the NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NNA at 50 microM). IJPs from electrical field stimulation triggered slow waves (TSWs); after TTX or omega-CTX, TSWs still occurred, advanced in time and increased in amplitude after TTX. Addition of L-NNA advanced the onset of the TSWs after omega-CTX. TTX, L-NNA, or omega-CTX left the resting membrane potentials, the characteristics of spontaneous slow waves, or TSWs evoked by a long stimulating pulse unchanged. L-NNA at 100 microM enhanced the amplitude but not the frequency of spontaneous slow waves. TTX and NOS blockers all increased circular muscle contractions associated with the spontaneous slow waves and TSWs. In isolated circular muscle preparations, the NOS inhibitors N omega-nitro-L-arginine methyl ester (L-NAME at 300 microM) or L-NNA at 100 microM abolished the IJPs and increased the regularity and amplitude of spontaneous slow waves and associated contractions, but TSWs could not be evoked before or after NOS inhibition. The NO donor 3-morpholinosydnonimine hydrochloride (SIN-1) at 200 microM caused hyperpolarizations (10-15 mV) similar to the IJP mediator, attenuated the IJPs, and abolished mechanical activities. SIN-1 increased the slow wave frequency but decreased the amplitude and duration of spontaneous slow waves and TSWs. VIP (10(-6) M) decreased contraction and slow wave amplitude and prolonged IJP duration without affecting membrane potential or slow wave frequency. We conclude that spontaneous slow waves and TSWs originate independently of neural activity. Pacemaking regions possess inhibitory neural inputs that release NO to mediate IJPs and relaxation and influence the delay before a TSW. NO (not VIP) release from nerves inhibits initiation of spontaneous slow waves or TSWs near the MyP, and spontaneous NO release modulates pacemaking activity from the DMP.

Heterogeneity in electrical activity of the canine ileal circular muscle: interaction of two pacemakers.

In cross-sectioned slabs from the muscularis externa or in the isolated circular muscle devoid of longitudinal muscle and myenteric plexus (MyP) of ileum, a slow wave, sigmoidal or triangular in shape, was recorded from microelectrode impalements near the deep muscular plexus (DMP) region in the whole-thickness preparation. From the MyP region of whole-thickness preparations, a slow wave which oscillated at a similar frequency (9-10 cycles min-1) was characterized by a fast upstroke and a square shape. Slow waves of mumixed pattern were recorded in the outer circular muscle (OCM) while triangular slow waves were present near the submucous plexus (SMP). In this preparation but not in isolated circular muscle, the inhibitory junction potentials (IJPs) produced by supramaximal electrical field stimulation triggered slow waves. The amplitudes (15-25 mV) of spontaneous and triggered slow waves (TSWs) were greatest in the MyP region, significantly so compared to those of DMP and SMP regions and to those in all regions of isolated circular muscle. Frequencies of slow waves recorded from the MyP and DMP were slightly but significantly higher than those recorded from either the OCM or the SMP or from all regions of isolated circular muscle. A 10-15 mV gradient in resting membrane potential (more hyperpolarized near MyP) existed across the intact (but not the isolated) circular muscle layer. Both types of slow waves, TSWs and IJPs were unaffected by muscarinic, adrenergic or tachykinergic blockade. We suggest that a MyP pacemaker network generated a plateau-type slow wave while a DMP one induced a triangular slow wave. Each source can function independently but the MyP network may dominate and entrain DMP slow waves.

Ca2+ role in myogenic and neurogenic activities of canine ileum circular muscle.

The role of Ca2+ in myogenic and neural activation in canine ileum circular muscle (CM) was studied during simultaneous recordings of contractile and electrical activity in cross-sectioned slabs of muscularis externa or of isolated CM with deep muscular plexus (DMP) intact. Ca(2+)-free Krebs solution abolished inhibitory junction potentials (IJP) and contractions before changes in CM membrane potentials and while slow waves (SW) persisted at lower amplitude and frequency. This medium abolished SW more rapidly in isolated CM than in intact muscle strips and affected triggered SW (TSW) by 100-ms pulses recorded near myenteric plexus or near DMP differentially in the full-thickness preparation; TSW did not occur in isolated CM. Ni2+, a nonselective Ca2+ channel antagonist, left IJP unchanged and reduced contractions, frequencies, and amplitudes of spontaneous SW and TSW, but increased their durations. Nifedipine abolished contractions but SW, TSW, and IJP were unaffected. Cyclopiazonic acid (CPA) increased SW frequency, produced spikes on SW plateaus, and increased CM tone, but did not affect IJP or resting membrane potentials. In nifedipine-pretreated strips, CPA decreased SW frequencies and amplitudes, evoked less tone, depolarized membrane potentials, and left IJP unaltered. The neuronal N-type Ca2+ channel blocker omega-conotoxin GVIA abolished IJP, without affecting SW or TSW. We conclude that Ca2+ influx, not through L- or N-type Ca2+ channels, helps initiate ileal SW; L-type Ca2+ channels provide Ca2+ for contraction and N-type Ca2+ channels provide Ca2+ for IJP mediator release. Frequencies of SW may be modulated by uptake of Ca2+ into pacemaker stores.

Role of sarcoplasmic reticulum in inhibitory junction potentials and hyperpolarizations by nitric oxide donors in opossum oesophagus.

1. Previous patch clamp studies of oesophageal circular muscle cells showed that nitric oxide (NO) modulated the opening of Ca2(+)-activated K+ channels involved in mediating the inhibitory junction potentials (i.j.ps). This study clarified the role of Ca2+ release from the superficial sarcoplasmic reticulum (SR) in the mechanism of i.j.ps or hyperpolarizing responses to NO-releasing compounds. Electrical and mechanical activities were simultaneously recorded by intracellular microelectrode or double sucrose gap techniques. 2. The NO-donors, sydnonimine (SIN-1) and sodium nitroprusside, each at 500 microM, hyperpolarized oesophageal circular muscle cells by 15-20 mV, like i.j.ps. 3. The selective inhibitors of SR Ca2(+)-ATPase (cyclopiazonic acid 10-30 microM and thapsigargin 5 microM) and the SR Ca2+ release channel activator (ryanodine 30 microM) caused depolarization and spontaneous contractions which were diminished after prolonged (> 30 min) incubation with these agents in Ca2(+)-containing medium. Moreover, these agents inhibited both the i.j.p. and NO-donor hyperpolarizations, suggesting that a functional SR Ca2+ uptake is necessary for the response to endogenous or exogenous NO. 4. These results, along with our previous findings of the dependence of i.j.ps and NO-donor hyperpolarizations on K+ channel activation and cyclic GMP elevation, support the hypothesis that subplasmalemmal (Ca2+)i elevation, via vectorial Ca2+ release from superficial SR toward the plasmalemma, may be an important mechanism by which NO, from NO-liberating compounds or released from inhibitory neurones induces relaxation and i.j.ps in opossum oesophagus.

K+ channel opening mediates hyperpolarizations by nitric oxide donors and IJPs in opossum esophagus.

The ionic mechanisms by which nitric oxide (NO) or a related compound mediates the inhibitory junction potentials (IJPs) of the opossum esophageal circular smooth muscle were studied using microelectrodes and double sucrose gap. The NO donors, 3-morpholino-sydnonimine hydrochloride and sodium nitroprusside, induced 15- to 20-mV hyperpolarizations that reversed near the potassium equilibrium potential as did the IJPs. They inhibited the IJPs and decreased electrotonic potentials (increased conductance) even during restoration of the resting membrane potential by application of depolarizing current. Quinine was more efficacious than apamin in inhibiting the IJPs or NO donor hyperpolarizations, whereas the other K+ channel blockers tested (tetraethylammonium, charybdotoxin, 4-aminopyridine, Cs+, and glibenclamide) were without effect. Glibenclamide abolished the hyperpolarizing effects of the K+ channel opener BRL-34915. Low Cl- Krebs (isethionate substitutions) caused hyperpolarizations, increased electrotonic potentials, and reduced IJPs. The neural blockers, tetrodotoxin, omega-conotoxin GVIA, and N omega-nitro-L-arginine methyl ester, inhibited IJPs but not the responses to NO donors, indicating a postjunctional effect. Methylene blue and cystamine, soluble guanylate cyclase inhibitors, suppressed IJPs and responses to NO donors. We conclude that NO mediates esophageal IJPs, which depend on guanosine 3',5'-cyclic monophosphate elevation and activation of quinine- and apamin-sensitive K+ channels.