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Complications associated with spinal cord injury (SCI) result from unregulated reflexes below the lesion level. Understanding neurotransmission distal to the SCI could improve quality of life by mitigating complications. The long-term impact of SCI on neurovascular transmission is poorly understood, but reduced sympathetic activity below the site of SCI enhances arterial neurotransmission (1). We studied sympathetic neurovascular transmission using a rat model of long-term paraplegia (T2–3) and tetraplegia (C6–7). Sixteen weeks after SCI, T2–3 and C6–7 rats had lower blood pressure (BP) than sham rats (103 ± 2 and 97 ± 4 vs. 117 ± 6 mmHg, P < 0.05). T2–3 rats had tachycardia (410 ± 6 beats/min), and C6–7 rats had bradycardia (299 ± 10 beats/min) compared with intact rats (321 ± 4 beats/min, P < 0.05). Purinergic excitatory junction potentials (EJPs) were measured in mesenteric arteries (MA) using microlectrodes, and norepinephrine (NE) release was measured using amperometry. NE release was similar in all groups, while EJP frequency-response curves from T2–3 and C6–7 rats were left-shifted vs. sham rats. EJPs in T2–3 and C6–7 rats showed facilitation followed by run-down during stimulation trains (10 Hz, 50 stimuli). MA reactivity to exogenous NE and ATP was similar in all rats. In T2–3 and C6–7 rats, NE content was increased in left cardiac ventricles compared with intact rats, but was not changed in MA, kidney, or spleen. Our data indicate that peripheral purinergic, but not adrenergic, neurotransmission increases following SCI via enhanced ATP release from periarterial nerves. Sympathetic BP support is reduced after SCI, but improving neurotransmitter release might maintain cardiovascular stability in individuals living with SCI. NEW & NOTEWORTHY This study revealed increased purinergic, but not noradrenergic, neurotransmission to mesenteric arteries in rats with spinal cord injury (SCI). An increased releasable pool of ATP in periarterial sympathetic nerves may contribute to autonomic dysreflexia following SCI, suggesting that purinergic neurotransmission may be a therapeutic target for maintaining stable blood pressure in individuals living with SCI. The selective increase in ATP release suggests that ATP and norepinephrine may be stored in separate synaptic vesicles in periarterial sympathetic varicosities.

The irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral sensation disorder that is more common in women than men. Female serotonin transporter (SERT)-gene knockout (KO) rats exhibit hypersensitivity to colorectal balloon distention (CRD) that mimics colonic hypersensitivity occurring in female IBS patients. Alosetron (5-HT3 receptor antagonist) is used to treat diarrhea-predominant IBS in female patients. Other 5-HT3 receptor antagonists are ineffective at treating IBS symptoms. The visceromotor response (VMR) to CRD in SERT-KO and wild-type (WT) rats was measured following subcutaneous (sc), intracerobroventricular (icv), or intrathecal (it) treatment with 5-HT3 receptor antagonists and an agonist. Alosetron (sc) and granisetron (antagonists) caused a paradoxical increase in the VMR to CRD in SERT-KO female rats. Alosetron (sc) increased the VMR to CRD in WT male rats. Alosetron (it) increased the VMR to CRD in SERT-KO female rats only, and the 5-HT3 receptor agonist SR-52772 increased the VMR to CRD in SERT-KO male rats. Depletion of spinal 5-HT using 5,7-dihydroxytryptamine prevented the increase in VMR to CRD in SERT-KO female and male rats treated it with alosetron and SR-52772, respectively. Alosetron (icv) did not affect the VMR to CRD in WT or KO female rats, but it increased the VMR in male SERT-KO but not WT male rats. These data suggest that 5-HT3 receptor signaling at the dorsal spinal cord mediates visceral hypersensitivity in female SERT-KO rats. Such differences could facilitate development of sex-specific drug treatments for visceral pain. NEW & NOTEWORTHY We studied a model of female sex-specific visceral hypersensitivity using rats that had a loss of function of the serotonin transporter (SERT) caused by gene truncation. Female SERT-KO rats exhibited visceral hypersensitivity in response to colorectal balloon distention. We found that increased 5-HT signaling at dorsal spine 5-HT3 receptors was responsible for visceral hypersensitivity in female but not male SERT-KO rats.

DOCA-salt and obesity-related hypertension are associated with inflammation and sympathetic nervous system hyperactivity. Prejunctional α2-adrenergic receptors (α2ARs) provide negative feedback to norepinephrine release from sympathetic nerves through inhibition of N-type Ca2+channels. Increased neuronal norepinephrine release in DOCA-salt and obesity-related hypertension occurs through impaired α2AR signaling; however, the mechanisms involved are unclear. Mesenteric arteries are resistance arteries that receive sympathetic innervation from the superior mesenteric and celiac ganglia (SMCG). We tested the hypothesis that macrophages impair α2AR-mediated inhibition of Ca2+channels in SMCG neurons from DOCA-salt and high-fat diet (HFD)-induced hypertensive rats. Whole cell patch-clamp methods were used to record Ca2+currents from SMCG neurons maintained in primary culture. We found that DOCA-salt, but not HFD-induced, hypertension caused macrophage accumulation in mesenteric arteries, increased SMCG mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α, and impaired α2AR-mediated inhibition of Ca2+currents in SMCG neurons. α2AR dysfunction did not involve changes in α2AR expression, desensitization, or downstream signaling factors. Oxidative stress impaired α2AR-mediated inhibition of Ca2+currents in SMCG neurons and resulted in receptor internalization in human embryonic kidney-293T cells. Systemic clodronate-induced macrophage depletion preserved α2AR function and lowered blood pressure in DOCA-salt rats. HFD caused hypertension without obesity in Sprague-Dawley rats and hypertension with obesity in Dahl salt-sensitive rats. HFD-induced hypertension was not associated with inflammation in SMCG and mesenteric arteries or α2AR dysfunction in SMCG neurons. These results suggest that macrophage-mediated α2AR dysfunction in the mesenteric circulation may only be relevant to mineralocorticoid-salt excess.NEW & NOTEWORTHY Here, we identify a contribution of macrophages to hypertension development through impaired α2-adrenergic receptor (α2AR)-mediated inhibition of sympathetic nerve terminal Ca2+channels in DOCA-salt hypertensive rats. Impaired α2AR function may involve oxidative stress-induced receptor internalization. α2AR dysfunction may be unique to mineralocorticoid-salt excess, as it does not occur in obesity-related hypertension.

We examined the impact of sex on high-fat diet (HFD)–induced renal alterations in Dahl salt-sensitive and Sprague Dawley rats. In Dahl rats, HFD (60% kcal from fat for 24–26 weeks starting at weaning) significantly and equally increased blood pressure in males and females when compared with rats fed a control diet (10% kcal from fat). Male Dahl rats on HFD exhibited progressive renal histological injury and moderately increased renal macrophage infiltration at 10 and 24 weeks of feeding when compared with males on control diet. Female Dahl rats had lower grade renal injury and less macrophage infiltration (except at 17 weeks) than males regardless of diet. Male Dahl rats on both diets showed progressively increasing numbers of renal T-cells, a pattern not observed in females. HFD per se did not significantly affect renal T-cell number. Male Dahl rats had lower renal regulatory T-cells cell ratio than females at 24 weeks. Renal macrophage and T-cell infiltrations were highly correlated to final mean arterial pressure levels in males but not in females. Sprague Dawley rats fed HFD were normotensive without significant renal injury/inflammation after 24 weeks of feeding. In summary, HFD feeding fails to increase arterial blood pressure in Sprague Dawley rats but strongly promotes hypertension in both male and female Dahl salt-sensitive rats. Only Dahl males, however, exhibited blood pressure-associated renal inflammation and injury. Maintenance of regulatory T-cells ratio may protect against hypertension-associated renal injury/inflammation but not HFD-induced hypertension.

Enteric inhibitory motoneurons use nitric oxide and a purine neurotransmitter to relax gastrointestinal smooth muscle. Enteric P/Q-type Ca2+ channels contribute to excitatory neuromuscular transmission; their contribution to inhibitory transmission is less clear. We used the colon from tottering mice ( tg/tg, loss of function mutation in the α1A pore-forming subunit of P/Q-type Ca2+ channels) to test the hypothesis that P/Q-type Ca2+ channels contribute to inhibitory neuromuscular transmission and colonic propulsive motility. Fecal pellet output in vivo and the colonic migrating motor complex (ex vivo) were measured. Neurogenic circular muscle relaxations and inhibitory junction potentials (IJPs) were also measured ex vivo. Colonic propulsive motility in vivo and ex vivo was impaired in tg/tg mice. IJPs were either unchanged or somewhat larger in tissues from tg/tg compared with wild-type (WT) mice. Nifedipine (L-type Ca2+ channel antagonist) inhibited IJPs by 35 and 14% in tissues from tg/tg and WT mice, respectively. The contribution of N- and R-type channels to neuromuscular transmission was larger in tissues from tg/tg compared with WT mice. The resting membrane potential of circular muscle cells was similar in tissues from tg/tg and WT mice. Neurogenic relaxations of circular muscle from tg/tg and WT mice were similar. These results demonstrate that a functional deficit in P/Q-type channels does not alter propulsive colonic motility. Myenteric neuron L-type Ca2+ channel function increases to compensate for loss of functional P/Q-type Ca2+ channels. This compensation maintains inhibitory neuromuscular transmission and normal colonic motility.

The spleen is a visceral organ that contracts during hypoxia to expel erythrocytes and immune cells into the circulation. Spleen contraction is under the control of noradrenergic sympathetic innervation. The activity of noradrenergic neurons terminating in the spleen capsule is regulated by α2-adrenergic receptors (AR). Interactions between endogenous cannabinoid signaling and noradrenergic signaling in other organ systems suggest endocannabinoids might also regulate spleen contraction. Spleens from mice congenitally lacking both CB1 and CB2 cannabinoid receptors (Cnr1 -/- /Cnr2 -/- mice) were used to explore the role of endocannabinoids in spleen contraction. Spleen contraction in response to exogenous norepinephrine (NE) was found to be significantly lower in Cnr1 -/- /Cnr2 -/- mouse spleens, likely due to decreased expression of capsular α1AR. The majority of splenic Cnr1 mRNA expression is by cells of the spleen capsule, suggestive of post-synaptic CB1 receptor signaling. Thus, these studies demonstrate a role for CB1 and/or CB2 in noradrenergic splenic contraction.

We have previously reported that high fat diet (HFD) with normal salt feeding strongly promotes hypertension in both male and female Dahl salt sensitive (SS) rats. Only males, however, exhibit hypertension-associated renal inflammation/injury. The underlying mechanisms responsible for sex differences in HFD associated renal inflammation/injury are undetermined. It has been reported that increased activity of Mammalian Target of Rapamycin Complex 1/2 (mTORC) contribute to high salt diet associated hypertension and renal inflammation/injury in Dahl SS male rats. In this study, we determined whether HFD increases renal mTORC signaling in Dahl SS rats and if there are sex differences in signaling. Renal cortex and medulla were collected from rats fed a control diet (CD, 10% kcal fat) or a HFD (60% kcal fat) with 0.3% NaCl for 10, 17 or 24 weeks (WKs, starting at weaning, the time points correspond to pre-hypertension, developing hypertension and established hypertension phases of the model. Ratio (%) of expression of phospho-S6 ribosomal protein Ser235/236 (pS6)/S6 (a marker for mTORC1), and phospho-AKT Ser473 (pAKT)/AKT (a marker for mTORC2) were used to evaluate mTORC activity. The activity of mTORC1 in CD male cortex and female medulla were slightly increased at 17 and 24WKs of feeding compared to 10 WKs (22±4 and 16±7 vs 2±2, P0.5). The overall activity of mTORC2 in CD males and females were very low and mostly unchanged during 24WKs feeding, except for a slight increase in CD male cortex at 17WKs of feeding compared to 10WKs (4±1 vs 0.1±0.02, P

Mesenteric arteries (MAs) are supplied densely by sympathetic nerves releasing NE and ATP to increase vascular tone. Increased neuronal ATP/NE storage and release and impaired clearance cause hypertension. Our previous studies of DOCA-salt hypertensive rats showed that 1) impairment of the prejunctional autoreceptors regulating ATP and NE release contributes to elevated sympathetic neurotransmission, and 2) purinergic neurotransmission was impaired due to decreased ATP bioavailability in sympathetic nerves. As hyperactivity of sympathetic nerves occurs in obesity-related hypertension, we investigated alterations in purinergic transmission in high fat-fed (HF) Dahl S rats. MAs maintained in vitro were stimulated focally. Intracellular recording of excitatory junction potential (EJP) from smooth muscle cells was obtained from male and female, control and HF rats. EJP amplitude, facilitation and rundown were assessed. EJPs from all groups were abolished by tetrodotoxin (300 nM), a Na + channel blocker, and P2X receptor antagonist, PPADS (10 μM), indicating EJPs were neurogenic and purinergic. At 17 weeks, we found that 1) frequency (0.5-10Hz) response curves from control and HF were similar in male and female arteries. 2) When using short trains of stimulation (0.5Hz; 5 pulses), UK14304 (0.0001-1μM)-, an α2-adrenergic receptor agonist, blocked EJPs response equally well in arteries from control and HF rats. Moreover, the α-adrenergic receptor antagonist, yohimbine (1 μM), potentiated EJPs similarly in control and HF groups. 3) There were no differences in EJP rundown caused by trains of stimulation (10Hz, 50 pulses) in MA from control and HF fed rats. Differential facilitation of purinergic neurotransmission was detected in male vs female. In the presence of yohimbine, degree of EJP facilitation was higher in female arteries compared to male control arteries (2.69±0.26 vs 1.96±0.09 N=4, p

Obesity hypertension is driven by sympathetic neurotransmission to the heart and blood vessels. We tested the hypothesis that high-fat diet (HFD)-induced hypertension is driven by sympathetic neurotransmission to mesenteric arteries (MA) in male but not female Dahl salt-sensitive (Dahl ss) rat. Rats were fed a control diet (CD; 10 kcal% from fat) or HFD (60 kcal% from fat) beginning at 3 weeks (wk) of age; measurements were made at 10-, 17- and 24-wk. Body weight increased with HFD, age and sex. Mean arterial pressure (MAP) was higher in HFD versus CD rats from both sexes at 17- and 24-wk. MA constriction measured using pressure myography, and electrical field stimulation (EFS, 0.2–30 Hz) was greater in HFD versus CD in males at 17-wk; this was not due to changes in α2 autoreceptor or norepinephrine transporter (NET) function. Prazosin (α1-AR antagonist) and suramin (P2 receptor antagonist) inhibited neurogenic MA constriction equally in all groups. Arterial reactivity to exogenous norepinephrine (NE; 10−8 - 10−5 M) was lower in HFD versus CD at 10-wk in males. Female MA reactivity to exogenous ATP was lower at 24-weeks compared to earlier time points. HFD did not affect tyrosine hydroxylase (TH) or the vesicular nucleotide transporter (VNUT) nerve density in MA from both sexes. NE content was lower in MA but higher in plasma at 24-wk compared to 10- and 17-wk in both sexes. In conclusion, HFD-induced hypertension is not driven by increased sympathetic neurotransmission to MA in male and female Dahl ss rats.

Obesity alters gastrointestinal (GI) motility and 5-HT signalling. Altered 5-HT signalling disrupts control of GI motility. Levels of extracellular 5-HT depend on enterochromaffin (EC) cell release and serotonin transporter (SERT) uptake. We assessed GI transit and 5-HT signalling in the jejunum of normal and obese mice. Male and female mice were fed a control diet (CD; 10% of kilocalories as fat) or a high-fat diet (HFD; 60% of kilocalories as fat). Gastrointestinal transit was increased in male HFD-fed and female CD-fed compared with male CD-fed mice. The 5-HT3 receptor blocker, alosetron, increased gastric emptying in male CD-fed mice, but decreased transit in female CD-fed mice. The 5-HT-induced jejunal longitudinal muscle contractions in vitro were similar in all mice. In contrast to male CD-fed mice, 5-HT uptake (measured using continuous amperometry in vitro) in male HFD-fed mice was fluoxetine insensitive, yet sensitive to cocaine and the dopamine transporter (DAT) blocker GBR 12909. Immunoreactivity for DAT was present in the mucosa, and protein levels were greater in male HFD-fed compared with CD-fed mice. Extracellular 5-HT and mucosal 5-hydroxyindolacetic acid (5-HT metabolite) were similar in male HFD-fed compared with CD-fed mice. 5-Hydroxytryptamine uptake was fluoxetine sensitive in all females. Greater SERT protein, decreased extracellular 5-HT and greater mucosal 5-hydroxyindolacetic acid were observed in female HFD-fed compared with CD-fed mice. Mucosal 5-HT and EC cell numbers were similar in CD-fed and HFD-fed mice of both sexes; female 5-HT and EC cell numbers were increased compared with males. The HFD did not alter plasma sex hormone levels in any mice. Overall, obesity alters GI transit and 5-HT signalling in a sex-dependent manner.

Opioid drugs have powerful antidiarrheal effects and many patients taking these drugs for chronic pain relief experience chronic constipation that can progress to opioid-induced bowel dysfunction. Three classes of opioid receptors are expressed by enteric neurons: μ-, δ-, and κ-opioid receptors (MOR, DOR, and KOR). MOR and DOR couple to inhibition of adenylate cylase and nerve terminal Ca(2+) channels and activation of K(+) channels. These effects reduce neuronal activity and neurotransmitter release. KOR couples to inhibition of Ca(2+) channels and inhibition of neurotransmitter release. In the human gastrointestinal tract, MOR, DOR, and KOR link to inhibition of acetylcholine release from enteric interneurons and purine/nitric oxide release from inhibitory motorneurons. These actions inhibit propulsive motility. MOR and DOR also link to inhibition of submucosal secretomotor neurons, reducing active Cl(-) secretion and passive water movement into the colonic lumen. These effects account for the constipation caused by opioid receptor agonists. Tolerance develops to the analgesic effects of opioid receptor agonists but not to the constipating actions. This may be due to differential β-arrestin-2-dependent opioid receptor desensitization and internalization in enteric nerves in the colon compared with the small intestine and in neuronal pain pathways. Further studies of differential opioid receptor desensitization and tolerance in subsets of enteric neurons may identify new drugs or other treatment strategies of opioid-induced bowel dysfunction.

Lysophospholipids (LysoPLs) are bioactive lipid species involved in cellular signaling processes and the regulation of cell membrane structure. LysoPLs are metabolized through the action of lysophospholipases, including lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2). A new X-ray crystal structure of LYPLA2 compared with a previously published structure of LYPLA1 demonstrated near-identical folding of the two enzymes; however, LYPLA1 and LYPLA2 have displayed distinct substrate specificities in recombinant enzyme assays. To determine how these in vitro substrate preferences translate into a relevant cellular setting and better understand the enzymes' role in LysoPL metabolism, CRISPR-Cas9 technology was utilized to generate stable KOs of

Increased sympathetic nervous system activity contributes to deoxycorticosterone acetate (DOCA)-salt hypertension in rats. ATP and norepinephrine (NE) are coreleased from perivascular sympathetic nerves. NE acts at prejunctional α 2 -adrenergic receptors ( α 2 ARs) to inhibit NE release, and α 2 AR function is impaired in DOCA-salt rats. Adenosine, an enzymatic ATP degradation product, acts at prejunctional A 1 adenosine receptors (A 1 Rs) to inhibit NE release. We tested the hypothesis that prejunctional A 1 R function is impaired in sympathetic nerves supplying mesenteric arteries (MAs) and veins (MVs) of DOCA-salt rats. Electrically evoked NE release and constrictions of blood vessels were studied in vitro with use of amperometry to measure NE oxidation currents and video microscopy, respectively. Immunohistochemical methods were used to localize tyrosine hydroxylase (TH) and A 1 Rs in perivascular sympathetic nerves. TH and A 1 Rs colocalized to perivascular sympathetic nerves. Adenosine and N 6 -cyclopentyl-adenosine (CPA, A 1 R agonist) constricted MVs but not MAs. Adenosine and CPA (0.001–10 µ M) inhibited neurogenic constrictions and NE release in MAs and MVs. DOCA-salt arteries were resistant to adenosine and CPA-mediated inhibition of NE release and constriction. The A 2A adenosine receptor agonist CGS21680 (C 23 H 29 N 7 O 6 .HCl.xH 2 O) (0.001–0.1 μ M) did not alter NE oxidation currents. We conclude that there are prejunctional A 1 Rs in arteries and both pre- and postjunctional A 1 Rs in veins; thus, adenosine selectively constricts the veins. Prejunctional A 1 R function is impaired in arteries, but not veins, from DOCA-salt rats. Sympathetic autoreceptor dysfunction is not specific to α 2 ARs, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release in DOCA-salt hypertension.

Inflammation and increased sympathetic activity contribute to hypertension. Alpha 2-adrenergic receptor (α2AR) activation decreases norepinephrine (NE) release from sympathetic nerves by inhibiting Ca 2+ channels. We hypothesized that macrophage infiltration into mesenteric arteries (MA) impairs α2AR in salt-sensitive and obesity-associated hypertensive rats. Uniphrectomized Sprague Dawley (SD) rats were given water (SHAM) or 200mg/kg DOCA (pellet, sc) and water containing 1% NaCl, 0.2% KCl (DOCA) for 4 weeks. A high fat diet (HFD, 60% kcal from fat, 0.33% NaCl, 1% K + ) or normal fat diet (NFD, 10% kcal from fat, 0.24% NaCl, 0.36% K + ) was given to a second group of SD rats for 20 weeks and to Dahl salt-sensitive (SS) rats for 24-26 weeks after weaning (3 weeks). Immunohistochemistry for CD163 (macrophage marker) was used to count macrophages in MA. Whole-cell patch clamp was used on dissociated celiac ganglion neurons to evaluate α2AR-mediated Ca 2+ current inhibition with NE (1 μM). Liposome-encapsulated clodronate (Clod) was used to deplete rats of macrophages. Plasma aldosterone levels were assessed by ELISA. Summary data is provided in Table 1. Systolic blood pressure was higher in DOCA vs SHAM and HFD vs NFD rats. Vascular macrophage number increased in DOCA vs SHAM but not in HFD vs NFD rats. NE inhibited Ca 2+ current to a greater degree in neurons of SHAM vs DOCA but not NFD vs HFD rats. Clodronate reduced vascular macrophages in all rats and preserved α2AR-mediated inhibition of Ca 2+ current in DOCA rats. HFD did not affect plasma aldosterone levels. Therefore, we conclude that macrophage-associated impairment of α2AR may only occur in states of mineralocorticoid and salt excess.

Obesity associated hypertension in rodent models is commonly associated with altered vascular reactivity to sympathetic neurotransmitters and inflammation-induced vascular remodeling/fibrosis. Dahl salt-sensitive (SS) rats exhibit elevated sympathetic activity and vascular remodeling. We hypothesized that diet-induced obesity in Dahl SS rats would promote hypertension, vascular dysfunction and remodeling/fibrosis. Male Dahl SS rats were placed on high fat diet (HFD, 60% kcal from fat with final concentrations of 0.33% NaCl and 1% K + , n=5) or normal-fat diet (NFD; 10% kcal from fat, 0.24% NaCl, 0.36% K + , n=5) for 24-26 weeks after weaning (3 weeks of age). Compared with NFD rats, HFD rats displayed severe hypertension (MAP, 165±4 mmHg vs 133±6 mmHg, P0.05). HFD did not affect pressurized mesenteric arterial (~300 μm inner diameter, 60 mmHg) reactivity to norepinephrine or ATP in vitro . Pressurized mesenteric arteries from HFD rats displayed thicker walls (Ca 2+ free buffer, 40±1 μm vs 36±1 μm, P

We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high-fat diet (HFD). We used wild-type (WT) mice and mice lacking the β1-subunit of the BK channel (BKβ1−/−). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons, and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-l-arginine (NLA) was less effective at inhibiting relaxations in HFD compared with control diet (CD) WT mice (11 vs. 37%, P < 0.05). SMCs from HFD WT mice had depolarized membrane potentials (−47 ± 2 mV) and continuous action potential firing compared with CD WT mice (−53 ± 2 mV, P < 0.05), which showed rhythmic firing. SMCs from HFD or CD fed BKβ1−/− mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BKβ1−/− mice. Migrating motor complexes were disrupted in colons from BKβ1−/− mice and HFD WT mice. BK channel α-subunit protein and β1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD-induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability, and colonic motility by promoting oxidative stress, loss of nitrergic neurons, and SMC BK channel dysfunction.

The transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated cation channel expressed by sensory nerves. P2Y receptors are G protein-coupled receptors that are also expressed by TRPV1-positive sensory neurons. Therefore, we studied interactions between P2Y receptors and TRPV1 function on kidney projecting sensory neurons. Application of Fast Blue (FB) to nerves surrounding the renal artery retrogradely labeled neurons in dorsal root ganglia of rats. Whole cell recording was performed on FB-labeled neurons maintained in primary culture. Capsaicin was used to activate TRPV1. Four types of kidney projecting neurons were identified based on capsaicin responses: 1) desensitizing (35%), 2) nondesensitizing (29%), 3) silent (3%), and 4) insensitive (30%). Silent neurons responded to capsaicin only after ATP (100 μM) pretreatment. ATP reversed desensitization in desensitizing neurons. Insensitive neurons never responded to capsaicin. UTP, a P2Y purinoceptor 2 (P2Y2)/P2Y4receptor agonist, reversed capsaicin-induced TRPV1 desensitization. 2-methyl-thio-ATP (2-Me-S-ATP), a P2Y1receptor agonist, did not change desensitization. MRS 2179 and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), drugs that block P2Y1receptors, did not block ATP-induced resensitization of TRPV1. Suramin, a P2Y2receptor antagonist, blocked resensitization caused by UTP. Immunocytochemical studies showed that FB-labeled neurons coexpressed P2Y2receptors and TRPV1. We conclude that P2Y2receptor activation can maintain TRPV1 function perhaps during sustained episodes of activity of kidney projecting sensory neurons.

The sympathetic nervous system is important in blood pressure regulation, and regionally-specific increases in sympathetic nerve activity occur during the development of hypertension. Sympathetic neurons innervating the splanchnic organs may be especially critical, because sympathetic activity to the splanchnic region is increased early in hypertension development. The celiac ganglionic plexus contains the majority of the sympathetic neurons innervating the splanchnic organs and tissues. Celiac ganglionectomy (CGX) involves surgical removal of the celiac ganglionic plexus, and has been used to study the roles of the splanchnic sympathetic innervation in cardiovascular regulation. In the current study we characterized the short-term (two-week) and long-term (five-week and ten-week) effects of CGX in rats on splanchnic sympathetic nerve structure and function. In the short-term, norepinephrine concentrations in whole splanchnic organs and mesenteric arteries and veins were significantly decreased by CGX. Immunohistochemistry and glyoxylic acid staining showed an almost complete loss of the typical sympathetic innervation of mesenteric arteries and veins. Additionally, CGRP-containing sensory nerves largely disappeared. Constrictor responses of mesenteric arteries and veins to sympathetic nerve stimulation were abolished by CGX. However, the effects of CGX were time-dependent, since significant regeneration of sympathetic nerves in some organs was observed 5weeks after surgery. The inferior mesenteric ganglion had minimal impact on this reinnervation process. In vivo studies showed that CGX significantly lowers resting blood pressure in normal Sprague-Dawley rats. Therefore, CGX is an effective means to impair sympathetic input to the splanchnic organs, but the effect of the procedure is not permanent.

We studied hypertension-associated changes in prejunctional alpha(2) adrenergic receptor (alpha(2)-AR) function using amperometry to monitor in vitro norepinephrine (NE) measured as oxidation currents. Vasoconstriction was measured using video imaging. NE release was induced by electrical stimulation of sympathetic nerves associated with mesenteric arteries (MA) and veins (MV) of sham and DOCA-salt hypertensive rats. NE oxidation currents were larger in DOCA-salt compared to sham MA; there were no differences between currents in sham and DOCA-salt MV. Increases in NE oxidation currents followed a multi-exponential time course in sham MA. In DOCA-salt MA and sham and DOCA-salt MV, the time course was mono-exponential. Yohimbine (alpha(2)-AR antagonist, 1 microM), caused a mono-exponential increase in NE oxidation currents in sham and DOCA-salt MA. Yohimbine increased NE oxidation currents and constrictions more in sham compared to DOCA-salt MA and compared to MV. UK 14,304 (alpha(2)-AR agonist, 1.0 microM), reduced currents less in DOCA-salt MA and sham and DOCA-salt MV compared to sham MA. Prazosin (alpha(1)-AR antagonist, 0.1 microM) did not alter NE oxidation currents. Prazosin inhibited constrictions more in DOCA-salt compared to sham MA and almost completely blocked constrictions in sham and DOCA-salt MV. Prazosin-resistant constrictions in MA were blocked by the P2 receptor antagonist, PPADS (10 microM). Prejunctional alpha(2)-ARs modify NE concentrations near neuroeffector junctions in MA and MV. alpha(2)-AR function is most prominent in MA and is impaired in DOCA-salt MA but not MV. Purinergic transmission predominates in sham MA. NE is the dominant vasoconstrictor in DOCA-salt MA and sham and DOCA-salt MV.

Neuromuscular transmission is crucial for normal gut motility but little is known about its postnatal maturation. This study investigated excitatory/inhibitory neuromuscular transmission in vitro using ileal nerve-muscle preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3-30 micromol L(-1)) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 micromol L(-1))-sensitive manner. The muscarinic receptor antagonist, scopolamine (1 micromol L(-1)), reduced substantially nicotine-induced contractions in neonatal tissues but not adult tissues. In the presence of N(omega)-nitro-l-arginine (NLA, 100 micromol L(-1)) to block nitric oxide (NO) mediated inhibitory neuromuscular transmission, scopolamine-resistant nicotine-induced contractions were revealed in neonatal tissues. NLA enhanced the nicotine-induced contractions in neonatal but not in adult tissues. Electrical field stimulation (20 V; 0.3 ms; 5-25 Hz, scopolamine 1 micromol L(-1) present) caused NLA and TTX-sensitive longitudinal muscle relaxations. Frequency-response curves in neonatal tissues were left-shifted compared with those obtained in adult tissues. Immunohistochemical studies revealed that NO synthase (NOS)-immunoreactivity (ir) was present in nerve fibres supplying the longitudinal muscle in neonatal and adult tissues. However, quantitative studies demonstrated that fluorescence intensity of NOS-ir nerve fibres was higher in neonatal than adult tissues. Nerve fibres containing substance P were abundant in longitudinal muscle in adult but not in neonatal tissues. Inhibitory neuromuscular transmission is relatively more effective in the neonatal guinea pig small intestine. Delayed maturation of excitatory motor pathways might contribute to paediatric motility disturbances.

Sympathetic nerves release norepinephrine and ATP onto mesenteric arteries. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, there is increased arterial sympathetic neurotransmission attributable, in part, to impaired prejunctional regulation of norepinephrine release. Prejunctional regulation purinergic transmission in hypertension is less well understood. We hypothesized that α 2 -adrenergic receptor dysfunction alters purinergic neurotransmission to arteries in DOCA-salt hypertensive rats. Mesenteric artery preparations were maintained in vitro, and intracellular electrophysiological methods were used to record excitatory junction potentials (EJPs) from smooth muscle cells. EJP amplitude was reduced in smooth muscle cells from DOCA-salt (4±1 mV) compared with control arteries (9±1 mV; P 2 adrenergic receptor antagonist yohimbine (1 μmol/L) potentiated EJPs in control more than in DOCA-salt arteries (180±35% versus 86±7%; P 2 adrenergic receptor agonist UK 14304 (0.001 to 0.100 μmol/L), the A 1 adenosine receptor agonist cyclopentyladensosine (0.3 to 100.0 μmol/L), and the N-type calcium channel blocker ω-conotoxin GVIA (0.0003 to 0.1000 μmol/L) decreased EJP amplitude equally well in control and DOCA-salt arteries. Trains of stimuli (10 Hz) depleted ATP stores more completely, and the latency to EJP recovery was longer in DOCA-salt compared with control arteries. These data indicate that there is reduced purinergic input to mesenteric arteries of DOCA-salt rats because of decreased ATP bioavailability in sympathetic nerves. These data highlight the potential importance of impaired purinergic regulation of arterial tone as a target for drug treatment of hypertension.

As arteries are resistance blood vessels while veins perform a capacitance function, it might be expected that sympathetic neural control of arteries and veins would differ. The function of sympathetic nerves supplying mesenteric arteries (MA) and veins (MV) in rats was investigated using in vitro continuous amperometry with a carbon fibre microelectrode and video imaging. We simultaneously measured noradrenaline (NA) overflow at the blood vessel adventitial surface and vasoconstriction evoked by electrical stimulation of perivascular sympathetic nerves. Sympathetic nerve arrangement was studied using glyoxylic acid-induced fluorescence of NA. We found that: (i) there were significant differences between MA and MV in the arrangement of sympathetic nerves; (ii) frequency–response curves for NA overflow and vasoconstriction for MV were left-shifted compared to MA; (iii) the P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μm), reduced constrictions in MA but not in MV while the α1-adrenergic receptor antagonist, prazosin (0.1 μm), blocked constrictions in MV but not in MA; (iv) NA overflow for MA was enhanced by the α2-adrenergic receptor antagonist, yohimbine (1.0 μm), and attenuated by the α2-adrenergic receptor agonist, UK 14,304 (1.0 μm), while yohimbine and UK 14,304 had little effect in MV; (v) cocaine (10 μm) produced larger increases in NA overflow in MA than in MV; (vi) UK 14,304 constricted MV but not MA while yohimbine reduced constrictions in MV but not MA. We conclude that there are fundamental differences in sympathetic neuroeffector mechanisms in MA and MV, which are likely to contribute to their different haemodynamic functions.

Gastrointestinal motility is controlled in part by the enteric nervous system (ENS) and by extrinsic sympathetic nerves. The ENS provides primary control over gut function while the sympathetic innervation modulates ENS activity (1). The myenteric plexus, a division of the ENS, is particularly important for control of gastrointestinal motility (2). Using electrophysiological criteria, myenteric neurons can be classified as S or AH neurons (3) and AH neurons may be intrinsic primary afferent neurons (4). In AH neurons the action potential has a Ca2+ shoulder that is dependent on activation of N-type Ca2+ channels (5). Ca2+ entering during the action potential triggers calcium release from intracellular stores (6,7). The rise in intracellular Ca2+ then activates a Ca2+-dependent K+ channel, causing a long-lasting afterhyperpolarization (AHP)(4,8,9). S neurons are interneurons and motorneurons (10). Although action potentials in S neurons are blocked completely by tetrodotoxin (sodium channel antagonist), they are also associated with increases in intracellular calcium that are partly dependent on activation of N-type Ca2+ channels (11). Guinea pig myenteric neurons express multiple Ca2+ subtypes (12). The total Ca2+current (ICa2+) in myenteric neurons is composed of contributions of Ca2+ influx through L- N- P/Q- and R-type Ca2+ channels (13-18). The R-type channels make the largest contribution to the total ICa2+ in myenteric neurons of guinea-pig small intestine maintained in the primary culture (13). R-type Ca2+ channels are composed of the pore forming α1E subunit (19,20). SNX 482 (21) or low concentrations of NiCl2 (13,22) can block selectively currents passing through R-type channels. Sympathetic nerves inhibit intestinal motility by releasing norepinephrine which activates inhibitory α2-adrenoceptors expressed by myenteric neurons and their nerve endings (24,25). α2-Adrenoceptors couple to inhibition of calcium influx through voltage-gated calcium channels via a G-protein dependent pathway in the nervous system (26-28). α2-Adrenoceptors couple to the inhibitory Go/Gi-protein to inhibit adenylyl cyclase or to interact directly with calcium channels (28). Coupling between α2-adrenoceptor and specific subtypes of voltage-gated calcium channels has not been studied myenteric neurons. This study was designed to identify the type of calcium channel that is inhibited following activation of the α2-adrenoceptor in myenteric neurons and in their varicosities. These studies were done using whole-cell patch clamp techniques and calcium imaging methods to study Ca2+ dynamics in guinea-pig small intestinal myenteric neurons maintained in primary culture.

The agonist actions of seven commercial neonicotinoid insecticides and nicotine were studied on nicotinic acetylcholine receptors (nAChRs) expressed by neurons isolated from the three thoracic ganglia of the American cockroach, Periplaneta americana. Single electrode voltage clamp recording was used to measure agonist-induced inward currents. Acetylcholine, nicotine and all neonicotinoids tested, except thiamethoxam, caused inward currents which were blocked reversibly by methyllycaconitine, a nAChR antagonist. Based on maximum inward currents, neonicotinoids could be divided into two subgroups: (1) those with a heterocyclic ring in their electronegative pharmacophore moiety (i.e. nicotine, imidacloprid and thiacloprid) were relatively weak partial agonists causing only 20-25% of the maximum ACh current and (2) open chain compounds (i.e. acetamiprid, dinotefuran, nitenpyram, and clothiandin) which were much more effective agonists producing 60-100% of the maximum ACh current. These compounds also elicited different symptoms of poisoning in American cockroaches with excitatory responses evident for the low efficacy agonists but depressive and paralytic responses predominating for the most efficacious agonists. No correlation was observed between agonist affinity and efficacy on these nAChRs. Thiamethoxam, even at 100 microM, failed to cause an inward current and showed no competitive interaction with other neonicotinoids on nAChRs, indicating that it is not a direct-acting agonist or antagonist. Despite the probable presence of multiple subtypes of nAChRs on cockroach neurons, competition studies between neonicotinoids did not reveal evidence that separate binding sites exist for the tested compounds. The size of inward currents induced by co-application of neonicotinoid pairs at equal concentration (100 microM) were predominantly determined by the one with higher binding affinity as indicated by EC(50) values, rather than by the one with higher binding efficacy as indicated by maximal current (I(max)). Agonist efficacy, but not affinity, was positively correlated with insecticidal activity. These findings indicate that: (1) agonist affinity and efficacy vary independently with neonicotinoid structure; (2) high agonist efficacy is dependent on the presence of an acyclic electronegative pharmacophore group; (3) agonist efficacy is a significant factor in the insecticidal activity of neonicotinoids to cockroaches; (4) lower efficacy compounds cause excitatory symptoms (Type A), while high efficacy compounds cause depressive/paralytic symptoms (Type B).

Background & Aims: 5-Hydroxytryptamine (5-HT) is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterochromaffin cells (EC), which release 5-HT, has not been studied in neonates. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs. Methods: 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and high-performance liquid chromatography with electrochemical detection. Results: An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface, and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 μM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs adult tissues. There was no difference in 5-HT content between neonates and adults but 5-hydroxyindole acetic acid/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number, but EC cells were found in the crypts in neonatal and along the villi in adult tissues. Conclusions: SERT expression is low in neonates, and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may be important for development of neurohumoral control of intestinal motor reflexes.

We tested the hypothesis that vascular macrophage infiltration and O2− release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3–5, 10–13, and 18–21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22 phox were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2− was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18–21. O2− and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22 phox were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2− while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2−, which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.

In sympathetic neurons, activation of the prejunctional alpha 2-adrenergic receptor (a2-AR) inhibits nerve terminal N-type Ca2+ channels and norepinephrine (NE) release onto blood vessels. In rats,...

Loss of endothelial cell-derived nitric oxide (NO) in hypertension is a hallmark of arterial dysfunction. Experimental hypertension created by the removal of NO, however, involves mechanisms in addition to decreased arterial vasodilator activity. These include augmented endothelin-1 (ET-1) release, increased sympathetic nervous system activity, and elevated tissue oxidative stress. We hypothesized that increased venous smooth muscle (venomotor) tone plays a role in Nomega-nitro-L-arginine (LNNA) hypertension through these mechanisms. Rats were treated with the NO synthase inhibitor LNNA (0.5 g/L in drinking water) for 2 weeks. Mean arterial pressure of conscious rats was 119 +/- 2 mm Hg in control and 194 +/- 5 mm Hg in LNNA rats (P

Superoxide anion (O2·−) production is elevated in the vasculature of hypertensive animals but it is not known if O2·−production is also elevated in the sympathetic nervous system. We measured O2·−levels in prevertebral sympathetic ganglia of deoxycorticosterone acetate (DOCA)-salt hypertensive rats using the dihydroethidine (DHE) fluorescence method. O2·−was elevated in ganglia from DOCA-salt rats compared with normotensive sham rats. Treatment of ganglia with endothelin (ET)-1 (3×10−8mol/L) resulted in a 200% increase in fluorescence intensity in neurons, which was attenuated by the ETBreceptor antagonist BQ788 (10−7mol/L). ET-1 also increased the O2·−induced fluorescence in dissociated sympathetic neurons and PC-12 cells via activation of ETBreceptors, but not ETAreceptors. To evaluate whether elevated ET-1 levels in the ganglia might contribute to the elevated O2·−found in ganglia we measured the amount of ET-1 using an ELISA assay. ET-1 levels in sham rat celiac ganglia were 695.6±40.9 picogram per gram; they were not different than ET-1 levels in ganglia from DOCA-salt rats. We then compared ETBreceptor levels in ganglia from sham and DOCA-salt animals. ETBreceptor mRNA levels were 32% higher and ETBreceptor protein levels were 20% higher in celiac ganglia from DOCA-salt rats than from sham rats separately. In conclusion, O2·−is elevated in prevertebral sympathetic ganglia in DOCA-salt hypertension, and ET-1 is a potent stimulus for the elevation of O2·−levels in sympathetic ganglia, an effect that may be mediated by the upregulation of ETBreceptors.

The present study tested the hypothesis that there is impaired function of α2-adrenergic autoreceptors and increased transmitter release from sympathetic nerves associated with mesenteric arteries and veins from DOCA-salt rats. High-performance liquid chromatography was used to measure the overflow of ATP and norepinephrine (NE) from electrically stimulated mesenteric artery and vein preparations in vitro. In sham arteries, nerve stimulation evoked a 1.5-fold increase in NE release, whereas in DOCA-salt arteries there was a 3.9-fold increase in NE release over basal levels ( P < 0.05). In contrast, stimulated ATP release was not different in DOCA-salt arteries compared with sham arteries. In sham veins, nerve stimulation evoked a 2.9-fold increase in NE release, whereas in DOCA-salt veins there was a 8.4-fold increase in NE release over basal levels ( P < 0.05). In sham rats NE release, normalized to basal levels, was greater in veins than in arteries ( P < 0.05). The α2-adrenergic receptor antagonist yohimbine (1 μM) increased ATP and NE release in sham but not DOCA-salt arteries. The α2-adrenergic receptor agonist UK-14,304 (10 μM) decreased ATP release in sham but not DOCA-salt arteries. In sham veins, UK-14,304 decreased, but yohimbine increased, NE release; effects that were not observed in DOCA-salt veins. These data show that nerve stimulation causes a greater increase in NE release from nerves associated with veins compared with arteries. In addition, impairment of α2-adrenergic autoreceptor function in sympathetic nerves associated with arteries and veins from DOCA-salt rats results in increased NE release.

P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. P2X receptors participate in intestinal neurotransmission but the subunit composition of enteric P2X receptors is unknown. In this study, we used tissues from P2X3 wild-type (P2X3+/+) mice and mice in which the P2X3 subunit gene had been deleted (P2X3-/-) to investigate the role of this subunit in neurotransmission in the intestine. RT-PCR analysis of mRNA from intestinal tissues verified P2X3 gene deletion. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from myenteric neurons in vitro. Drug-induced longitudinal muscle contractions were studied in vitro. Intraluminal pressure-induced reflex contractions (peristalsis) of ileal segments were studied in vitro using a modified Trendelenburg preparation. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. Fast excitatory postsynaptic potentials recorded from S neurons (motoneurons and interneurons) were similar in tissues from P2X3+/+ and P2X3-/- mice. S neurons from P2X3+/+ and P2X3-/- mice were depolarized by application of ATP but not alpha,beta-methylene ATP, an agonist of P2X3 subunit-containing receptors. ATP and alpha,beta-methylene ATP induced depolarization of AH (sensory) neurons from P2X3+/+ mice. ATP, but not alpha,beta-methylene ATP, caused depolarization of AH neurons from P2X3-/- mice. Peristalsis was inhibited in ileal segments from P2X3-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X3+/+ and P2X3-/- mice. Gastrointestinal transit was similar in P2X3+/+ and P2X3-/- mice. It is concluded that P2X3 subunit-containing receptors participate in neural pathways underlying peristalsis in the mouse intestine in vitro. P2X3 subunits are localized to AH (sensory) but not S neurons. P2X3 receptors may contribute to detection of distention or intraluminal pressure increases and initiation of reflex contractions.

Deoxycorticosterone acetate (DOCA) salt hypertension is associated with an endothelin-1 (ET-1)-dependent increase in arterial resistance and mean circulatory filling pressure. Contraction of endothelium-intact arteries and veins from sham and DOCA-salt hypertensive rats to agonists of the ETA (ET-1(1–31)) and ETB receptor (sarafotoxin 6c; S6c) was investigated in tissue baths as was expression of mRNA for ET-1 and mRNA and protein for the ETA and ETB receptor. ET-1(1–31) contracted aorta and vena cava from sham rats with a 30 fold lower potency than ET-1. Contraction was not altered by the ETB receptor antagonist BQ788 (100 nM) but was abolished by the ETA receptor antagonist ABT-627 (30 nM). In DOCA-salt thoracic aorta, maximum contraction to ET-1 and ET-1(1–31) was reduced (36.6±6.3 and 13.3±4.4% of sham response, respectively); aorta did not contract to S6c. In vena cava from DOCA-salt rats, contraction to ET-1 and ET-1(1–31) was not reduced compared to sham contraction; vena cava from sham and DOCA-salt rats contracted to S6c with a similar potency. Real time RT–PCR revealed that prepro ET-1 mRNA was increased 6.6±3.3 fold and 8.7±3.9 fold greater in DOCA-salt aorta and vena cava, respectively, compared to sham. Vena cava expressed a higher content of ETA and ETB receptor mRNA than aorta (P

We determined the contribution of vascular large conductance Ca2+-activated K+(BK) and L-type Ca2+channel dysregulation to exaggerated mortality in cecal ligation/puncture (CLP)-induced septic BK channel β1-subunit knockout (BK β1-KO, smooth muscle specific) mice. CLP-induced hemodynamic changes and mortality were assessed over 7 days in wild-type (WT) and BK β1-KO mice that were either untreated, given volume resuscitation (saline), or saline + calcium channel blocker nicardipine. Some mice were euthanized 24 h post-CLP to measure tissue injury and vascular and immune responses. CLP-induced hypotension was similar in untreated WT and BK β1-KO mice, but BK β1-KO mice died sooner. At 24 h post-CLP (mortality latency in BK β1-KO mice), untreated CLP-BK β1-KO mice showed more severe hypothermia, lower tissue perfusion, polymorphonuclear neutrophil infiltration-independent severe intestinal necrosis, and higher serum cytokine levels than CLP-WT mice. Saline resuscitation improved survival in CLP-WT but not CLP-BK β1-KO mice. Saline + nicardipine-treated CLP-BK β1-KO mice exhibited longer survival times, higher tissue perfusion, less intestinal injury, and lower cytokines versus untreated CLP-BK β1-KO mice. These improvements were absent in treated CLP-WT mice, although saline + nicardipine improved blood pressure similarly in both septic mice. At 24 h post-CLP, BK and L-type Ca2+channel functions in vitro were maintained in mesenteric arteries from WT mice. Mesenteric arteries from BK β1-KO mice had blunted BK/enhanced L-type Ca2+channel function. We conclude that vascular BK channel deficiency exaggerates mortality in septic BK β1-KO mice by activating L-type Ca2+channels leading to blood pressure-independent tissue ischemia.

BK channels regulate arterial tone. Studies done in humans and animals suggest that impaired BK channel function contributes to obesity associated hypertension due to higher myogenic tone and increased norepinephrine (NE) reactivity. In current study, we continually measured mean arterial pressure (MAP) and heart rate (HR) using radiotelemetry in high fat (HF) diet fed wild type (WT) and BK channel β1-subunit knockout (BK β1-KO) mice during last 11 weeks of HF-fed. After 24 weeks on HF-fed, body weight and blood glucose levels were similar in all mice. All mice were normotensive, but BK β1-KO mice showed a progressive fall in HR when compared to WT mice. Atenolol (oral) or hexamethonium (ip) treatment reduced MAP and HR similarly in all mice, prazosin (oral) decreased MAP and increased HR in BK β1-KO only. Mesenteric arteries (MA) from BK β1-KO mice exhibited increased sensitivity to NE and BayK 8644, higher myogenic tone, increased wall thickness and smaller wall/lumen ratio compared to WT mice. BK β1-KO...

These studies were carried out to characterize the properties of gamma-aminobutyric acidA (GABA(A)) receptors on guinea pig intestinal myenteric neurons maintained in primary culture. In addition, the type of neuron expressing GABA(A) receptors was identified using immunohistochemical methods. Whole-cell patch clamp recordings of currents elicited by GABA and acetylcholine (ACh) were obtained using pipettes containing Neurobiotin. After electrophysiological studies, neurons were processed for localization of calbindin-D28K-immunoreactivity (calbindin-ir). GABA (1 mM) and ACh (3 mM) caused inward currents in most cells tested. GABA currents were mimicked by muscimol (1-300 microM) and were blocked by bicuculline (10 microM) indicating that GABA was acting at GABA(A) receptors. GABA currents were associated with a conductance increase and a linear current/voltage relationship with a reversal potential of 1 +/- 1 mV (n = 5). Pentobarbital (PB, 3-1000 microM) and diazepam (DZP, 0.01-10 microM) potentiated GABA-induced currents. A maximum concentration of DZP (1 microM) increased GABA-induced currents 3.1 +/- 0.3 times while PB (1000 microM) increased GABA currents by 11 +/- 2 times. In outside-out patches, the amplitude of GABA-activated single-channel currents was linearly related to membrane potential with a single-channel conductance of 28.5 + 0.5 pS (n = 10). PB and DZP increased the open probability of GABA-induced single-channel currents. Neurons containing calbindin-ir were large, were isolated from other neurons and had GABA current amplitudes of -3.4 +/- 0.3 nA (n = 48). Neurons with weak or absent calbindin-ir were smaller, were localized in clusters of cells and had GABA-induced current amplitudes of -0.6 +/- 0.1 nA (n = 20). ACh-induced currents were smaller in calbindin-ir neurons (-0.7 +/- 0.1 nA) compared to weakly calbindin-ir neurons (-1.4 +/- 0.1 nA). These results indicate that myenteric calbindin-ir neurons express a high density of GABA(A) receptors. Cell size and location allow visual identification of neurons likely to contain calbindin-ir permitting targeted studies of the properties of these neurons.

The mechanism of catecholamine release from single adrenal chromaffin cells isolated from normotensive and DOCA-salt hypertensive rats was investigated. These cells were used as a model for sympathetic nerves to better understand how exocytotic release of catecholamines is altered in this model of hypertension. Catecholamine secretion was evoked by local application of acetylcholine (1 mM) or high K+ (70 mM), and continuous amperometry was used to monitor catecholamine secretion as an oxidative current. The total number of catecholamine molecules secreted from a vesicle, the total number of vesicles fusing and secreting, and the duration of secretion in response to a stimulus were all significantly greater for chromaffin cells from hypertensive rats as compared to normotensive controls. The greater catecholamine secretion from DOCA-salt cells results, at least in part, from functionally impaired large conductance, Ca2+-activated (BK) and ATP-sensitive K+ channels. This work reveals that there is altered vesicular release of catecholamines from these cells (and possibly from perivascular sympathetic nerves) and this may contribute to increased vasomotor tone in DOCA-salt hypertension.