Zinc drives vasorelaxation by acting in sensory nerves, endothelium and smooth muscle.

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

Early urinary tract infection after spinal cord injury: a retrospective inpatient cohort study.

STUDY DESIGN: Retrospective audit. OBJECTIVES: Examine factors associated with urinary tract infection (UTI), UTI incidence and impact on hospital length of stay (LOS) in new, inpatient adult traumatic spinal cord injury (SCI). SETTING: Western Australian Hospitals managing SCI patients. METHODS: Data on UTIs, bladder management and LOS were obtained from hospital databases and medical records over 26 months. Adherence to staff-administered intermittent catheterisation (staff-IC) was determined from fluid balance charts. RESULTS: Across the cohort (n = 70) UTI rate was 1.1 starts/100 days; UTI by multi-resistant organisms 0.1/100 days. Having ≥1 UTIs compared with none and longer duration of initial urethral indwelling catheterisation (IDC) were associated with longer LOS (p-values < 0.001). For patients with ≥1 UTIs (n = 43/70), longer duration of initial IDC was associated with shorter time to first UTI (1 standard deviation longer [SD, 45.0 days], hazard ratio (HR): 0.7, 95% confidence interval [CI] 0.5-1.0, p-value 0.044). In turn, shorter time to first UTI was associated with higher UTI rate (1 SD shorter [30.7 days], rate ratio (RR): 1.32, 95%CI 1.0-1.7, p-value 0.039). During staff-IC periods (n = 38/70), protocols were followed (85.7% ≤ 6 h apart, 96.1% < 8 h), but 26% of IC volumes exceeded 500 mL; occasional volumes > 800 mL and interruptions requiring temporary IDC were associated with higher UTI rates the following week (odds ratios (ORs): 1.6, 95%CI 1.1-2.3, p-value 0.009; and 3.9, 95%CI 2.6-5.9, p-value < 0.001 respectively). CONCLUSIONS: Reducing initial IDC duration and limiting staff-IC volumes could be investigated to possibly reduce inpatient UTIs and LOS. SPONSORSHIP: None.

The Effects of Diabetes and High-Fat Diet on Polymodal Nociceptor and Cold Thermoreceptor Nerve Terminal Endings in the Corneal Epithelium.

Purpose: There is a substantial body of evidence indicating that corneal sensory innervation is affected by pathology in a range of diseases. However, there are no published studies that have directly assessed whether the nerve fiber density of the different subpopulations of corneal sensory neurons are differentially affected. The present study explored the possibility that the intraepithelial nerve fiber density of corneal polymodal nociceptors and cold thermoreceptors are differentially affected in mice fed with a high-fat high cholesterol (HFHC; 21% fat, 2% cholesterol) diet and in those that also have diabetes. Methods: The mice were fed the HFHC diet for the duration of the experiment (up to 40 weeks). Mice in the diabetes group had hyperglycaemia induced with streptozotocin after 15 weeks on the HFHC diet. Age-matched control animals were fed a standard diet. All corneal nerve fibers were labeled with a pan neuronal antibody (antiprotein gene product 9.5), and polymodal nociceptors and cold thermoreceptors were labeled with antibodies directed against transient receptor potential cation channel, subfamily V, member 1 and transient receptor potential cation channel subfamily M member 8, respectively. Results: The mice fed a HFHC diet and those that in addition have hyperglycemia have similar reductions in corneal nerve fiber density consistent with small fiber neuropathy. Importantly, both treatments more markedly affected the intraepithelial axons of cold thermoreceptors than those of polymodal nociceptors. Conclusions: The results provide evidence that distinct subpopulations of corneal sensory neurons can be differentially affected by pathology.

The neurochemistry and morphology of functionally identified corneal polymodal nociceptors and cold thermoreceptors.

It is generally believed that the unencapsulated sensory nerve terminals of modality specific C- and Aδ-neurons lack structural specialization. Here we determined the morphology of functionally defined polymodal receptors and cold thermoreceptors in the guinea pig corneal epithelium. Polymodal receptors and cold thermoreceptors were identified by extracellular recording at the surface of the corneal epithelium. After marking the recording sites, corneas were processed to reveal immunoreactivity for the transient receptor potential channels TRPV1 (transient receptor potential cation channel, subfamily V, member 1) or TPRM8 (transient receptor potential cation channel subfamily M member 8). Polymodal receptor nerve terminals (n = 6) were TRPV1-immunoreactive and derived from an axon that ascended from the sub-basal plexus to the squamous cell layer where it branched into fibers that ran parallel to the corneal surface and terminated with small bulbar endings (ramifying endings). Cold thermoreceptor nerve terminals were TRPM8-immunoreactive (n = 6) and originated from an axon that branched as it ascended through the wing cell and squamous cell layers and terminated with large bulbar endings (complex endings). These findings indicate that modality specific corneal sensory neurons with unencapsulated nerve endings have distinct nerve terminal morphologies that are likely to relate to their function.

Changes in sympathetic neurovascular function following spinal cord injury.

The effects of spinal cord injury (SCI) on sympathetic neurovascular transmission have generally been ignored. This review describes changes in sympathetic nerve-mediated activation of arterial vessels to which ongoing sympathetic activity has been reduced or silenced following spinal cord transection in rats. In all vessels studied in rats, SCI markedly enhanced their contractile responses to nerve activity. However, the mechanisms that augment neurovascular transmission differ between the rat tail artery and mesenteric artery. In tail artery, the enhancement of neurovascular transmission cannot be attributed to changes in sensitivity of the vascular muscle to α1- or α2-adrenoceptor agonists. Instead the contribution of L-type Ca2+ channels to activation of the smooth muscle by nerve-released noradrenaline is greatly increased following SCI. By contrast, mesenteric arteries from SCI rats had increased sensitivity to phenylephrine but not to methoxamine. While both phenylephrine and methoxamine are α1-adrenoceptor agonists, only phenylephrine is a substrate for the neuronal noradrenaline transporter. Therefore the selective increase in sensitivity to phenylephrine suggests that the activity of the neuronal noradrenaline transporter is reduced. While present evidence suggests that sympathetic vasoconstrictor neurons do not contribute to the normal regulation of peripheral resistance below a complete SCI in humans, the available evidence does indicate that these experimental findings in animals are likely to apply after SCI in humans and contribute to autonomic dysreflexia.

TFOS DEWS II pain and sensation report.

Pain associated with mechanical, chemical, and thermal heat stimulation of the ocular surface is mediated by trigeminal ganglion neurons, while cold thermoreceptors detect wetness and reflexly maintain basal tear production and blinking rate. These neurons project into two regions of the trigeminal brain stem nuclear complex: ViVc, activated by changes in the moisture of the ocular surface and VcC1, mediating sensory-discriminative aspects of ocular pain and reflex blinking. ViVc ocular neurons project to brain regions that control lacrimation and spontaneous blinking and to the sensory thalamus. Secretion of the main lacrimal gland is regulated dominantly by autonomic parasympathetic nerves, reflexly activated by eye surface sensory nerves. These also evoke goblet cell secretion through unidentified efferent fibers. Neural pathways involved in the regulation of meibomian gland secretion or mucin release have not been identified. In dry eye disease, reduced tear secretion leads to inflammation and peripheral nerve damage. Inflammation causes sensitization of polymodal and mechano-nociceptor nerve endings and an abnormal increase in cold thermoreceptor activity, altogether evoking dryness sensations and pain. Long-term inflammation and nerve injury alter gene expression of ion channels and receptors at terminals and cell bodies of trigeminal ganglion and brainstem neurons, changing their excitability, connectivity and impulse firing. Perpetuation of molecular, structural and functional disturbances in ocular sensory pathways ultimately leads to dysestesias and neuropathic pain referred to the eye surface. Pain can be assessed with a variety of questionaires while the status of corneal nerves is evaluated with esthesiometry and with in vivo confocal microscopy.

Spinal cord thermosensitivity: An afferent phenomenon?

We review the evidence for thermoregulatory temperature sensors in the mammalian spinal cord and reach the following conclusions. 1) Spinal cord temperature contributes physiologically to temperature regulation. 2) Parallel anterolateral ascending pathways transmit signals from spinal cooling and spinal warming: they overlap with the respective axon pathways of the dorsal horn neurons that are driven by peripheral cold- and warm-sensitive afferents. 3) We hypothesize that these 'cold' and 'warm' ascending pathways transmit all extracranial thermosensory information to the brain. 4) Cutaneous cold afferents can be activated not only by cooling the skin but also by cooling sites along their axons: we consider that this is functionally insignificant in vivo. 5) By a presynaptic action on their central terminals, local spinal cooling enhances neurotransmission from incoming 'cold' afferent action potentials to second order neurons in the dorsal horn; this effect disappears when the spinal cord is warm. 6) Spinal warm sensitivity is due to warm-sensitive miniature vesicular transmitter release from afferent terminals in the dorsal horn: this effect is powerful enough to excite second order neurons in the 'warm' pathway independently of any incoming sensory traffic. 7) Distinct but related presynaptic mechanisms at cold- and warm-sensitive afferent terminals can thus account for the thermoregulatory actions of spinal cord temperature.

Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations.

The cornea is innervated by three main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1) expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion (TG) and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors) and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons). There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into three subpopulations on the basis of calcitonin gene-related peptide (CGRP) and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90%) are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal epithelium.

Angiotensin II increases nerve-evoked contractions in mouse tail artery by a T-type Ca(2+) channel-dependent mechanism.

Angiotensin II (Ang II) increases sympathetic nerve-evoked contractions of arterial vessels. Here the mechanisms underlying this effect were investigated in mouse tail artery. Isometrically mounted segments of mouse distal tail artery were used to investigate the effects of endothelium denudation, blocking Ca(2+) channels and inhibiting superoxide signalling on Ang II-induced facilitation of nerve-evoked contractions. In addition, in situ amperometry was used to assess effects of Ang II on noradrenaline release. Ang II (0.1-1nM) increased nerve-evoked contractions but did not change noradrenaline release. Losartan (Ang II type 1 receptor antagonist), but not PD 123319 (Ang II type 2 receptor antagonist), blocked the facilitatory effect of Ang II on nerve-evoked contractions. Ang II increased vascular muscle reactivity to phenylephrine and UK-14304 (α1- and α2-adrenoceptor agonists, respectively). Endothelial denudation increased nerve-evoked contractions and reduced the facilitatory effect of Ang II on these responses. Efonidipine (L- and T-type Ca(2+) channel blocker) and NNC 55-0396 (T-type Ca(2+) channel blocker) also attenuated this effect of Ang II, while nifedipine (L-type Ca(2+) channel blocker) did not. Blockers of superoxide generation/signalling did not change the facilitatory effect of Ang II on nerve-evoked contractions. The findings indicate that Ang II increases the contribution of T-type Ca(2+) channels to neural activation of the vascular muscle. In addition, Ang II appears to reduce the inhibitory influence of the endothelium on nerve-evoked contractions.

Analysis of the ghrelin receptor-independent vascular actions of ulimorelin.

Ulimorelin (TZP101) is a ghrelin receptor agonist that stimulates intestinal motility, but also reduces blood pressure in rodents and humans and dilates blood vessels. It has been proposed as a treatment for intestinal motility disorders. Here we investigated the mechanisms through which ulimorelin affects vascular diameter. Actions of ulimorelin on wall tension of rodent arteries were investigated and compared with other ghrelin receptor agonists. Saphenous, mesenteric and basilar arteries were obtained from Sprague-Dawley rats (male, 8 weeks) and saphenous arteries were obtained from wild type or ghrelin receptor null mice. These were mounted in myography chambers to record artery wall tension. Ulimorelin (0.03-30µM) inhibited phenylephrine-induced contractions of rat saphenous (IC50=0.6µM; Imax=66±5%; n=3-6) and mesenteric arteries (IC50=5µM, Imax=113±16%; n=3-4), but not those contracted by U46619, ET-1 or 60mM [K(+)]. Relaxation of phenylephrine-constricted arteries was not observed with ghrelin receptor agonists TZP102, capromorelin or AZP-531. In rat saphenous and basilar arteries, ulimorelin (10-100µM) and TZP102 (10-100µM) constricted arteries (EC50=9.9µM; Emax=50±7% and EC50=8µM; Emax=99±16% respectively), an effect not attenuated by the ghrelin receptor antagonist YIL 781 3µM or mimicked by capromorelin or AZP-531. In mesenteric arteries, ulimorelin, 1-10µM, caused a surmountable rightward shift in the response to phenylephrine (0.01-1000µM; pA2=5.7; n=3-4). Ulimorelin had similar actions in mouse saphenous artery from both wild type and ghrelin receptor null mice. We conclude that ulimorelin causes vasorelaxation through competitive antagonist action at α1-adrenoceptors and a constrictor action not mediated via the ghrelin receptor.

Modified cytoplasmic Ca2+ sequestration contributes to spinal cord injury-induced augmentation of nerve-evoked contractions in the rat tail artery.

In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.

Increased peripherin in sympathetic axons innervating plantar metatarsal arteries in STZ-induced type I diabetic rats.

A common characteristic of axonopathy is the abnormal accumulation of cytoskeletal proteins. We recently reported that streptozotocin (STZ)-induced type 1 diabetes produced a change in the morphology of sympathetic nerve fibers supplying rat plantar metatarsal arteries (PMAs). Here we investigated whether these morphological changes are associated with axonal accumulation of the type III intermediate filament peripherin and the microtubule protein ß-tubulin III, as both are implicated in axonal remodeling. PMAs from hyperglycemic STZ-treated rats receiving a low dose of insulin (STZ-LI) were compared with those from normoglycemic STZ-treated rats receiving a high dose of insulin (STZ-HI) and vehicle-treated controls. Western blotting revealed an increase in protein expression level for peripherin in PMAs from STZ-LI rats but no change in that for ß-tubulin III. In addition, there was an increase in the number of peripherin immunoreactive nerve fibers in the perivascular nerve plexus of PMAs from STZ-LI rats. Co-labeling for peripherin and neuropeptide Y (a marker for sympathetic axons) revealed that peripherin immunoreactivity increased in sympathetic axons. None of these changes were detected in PMAs from STZ-HI rats, indicating that increased peripherin in sympathetic axons of STZ-LI rats is likely due to hyperglycemia and provides a marker of diabetes-induced nerve damage.

Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor.

BACKGROUND AND PURPOSE: Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH: The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS: Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS: Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin.

Piezo2 expression in corneal afferent neurons.

Recently, a novel class of mechanically sensitive channels has been identified and have been called Piezo channels. In this study, we explored Piezo channel expression in sensory neurons supplying the guinea pig corneal epithelium, which have well-defined modalities in this species. We hypothesized that a proportion of corneal afferent neurons express Piezo2, and that these neurons are neurochemically distinct from corneal polymodal nociceptors or cold-sensing neurons. We used a combination of retrograde tracing to identify corneal afferent neurons and double label in situ hybridization and/or immunohistochemistry to determine their molecular and/or neurochemical profile. We found that Piezo2 expression occurs in ∼26% of trigeminal ganglion neurons and 30% of corneal afferent neurons. Piezo2 corneal afferent neurons are almost exclusively non-calcitonin gene-related peptide (CGRP)-immunoreactive (-IR), medium- to large-sized neurons that are NF200-IR, suggesting they are not corneal polymodal nociceptors. There was no coexpression of Piezo2 and transient receptor potential cation channel subfamily M member 8 (TRPM8) transcripts in any corneal afferent neurons, further suggesting that Piezo2 is not expressed in corneal cold-sensing neurons. We also noted that TRPM8-IR or CGRP-IR corneal afferent neurons are almost entirely small and lack NF200-IR. Piezo2 expression occurs in a neurochemically distinct subpopulation of corneal afferent neurons that are not polymodal nociceptors or cold-sensing neurons, and is likely confined to a subpopulation of pure mechano-nociceptors in the cornea. This provides the first evidence in an in vivo system that Piezo2 is a strong candidate for a channel that transduces noxious mechanical stimuli.

Spinal cord injury increases the reactivity of rat tail artery to angiotensin II.

Studies in individuals with spinal cord injury (SCI) suggest the vasculature is hyperreactive to angiotensin II (Ang II). In the present study, the effects of SCI on the reactivity of the rat tail and mesenteric arteries to Ang II have been investigated. In addition, the effects of SCI on the facilitatory action of Ang II on nerve-evoked contractions of these vessels were determined. Isometric contractions of artery segments from T11 (tail artery) or T4 (mesenteric arteries) spinal cord-transected rats and sham-operated rats were compared 6-7 weeks postoperatively. In both tail and mesenteric arteries, SCI increased nerve-evoked contractions. In tail arteries, SCI also greatly increased Ang II-evoked contractions and the facilitatory effect of Ang II on nerve-evoked contractions. By contrast, SCI did not detectably change the responses of mesenteric arteries to Ang II. These findings provide the first direct evidence that SCI increases the reactivity of arterial vessels to Ang II. In addition, in tail artery, the findings indicate that Ang II may contribute to modifying their responses following SCI.

Removal of half the sympathetic innervation does not reduce vasoconstrictor responses in rat tail artery.

Following reinnervation of denervated rat tail arteries, nerve-evoked contractions are at least as large as those evoked in normally innervated arteries despite a much lower nerve terminal density. Here nerve-evoked contractions have been investigated after transection of half the sympathetic innervation of normal tail arteries. After 1 week, the noradrenergic plexus 50-70 mm along the tail was about half as dense as control. Excitatory junction potentials recorded in smooth muscle cells of arterial segments isolated in vitro were half their normal amplitude. Surprisingly, nerve-evoked contractions of isometrically mounted segments were not reduced in amplitude, as was also the case after only 3 days. After 1 week, enhancement of nerve-evoked contractions by blocking either neuronal re-uptake of noradrenaline with desmethylimipramine or prejunctional α2-adrenoceptors with idazoxan was similar to control, suggesting that these mechanisms are matched to the number of innervating axons. The relative contribution of postjunctional α2-adrenoceptors to contractions evoked by long trains of stimuli was enhanced but that of α1-adrenoceptors was unchanged. Transiently, sensitivity to the α1-adrenoceptor agonist phenylephrine was slightly increased. After 7 weeks, amplitudes of nerve-evoked contractions remained similar to control, and sensitivity to phenylephrine had recovered but that to the α2-adrenoceptor agonist clonidine was slightly raised. The normal amplitude of nerve-evoked contractions after partial denervation is only partly explained by the greater contribution of α2-adrenoceptors. While the post-receptor mechanisms activated by nerve-released transmitter may be modified to amplify the contractions after partial denervation, our findings suggest that these mechanisms are normally saturated, at least in this artery.

Sensory and sympathetic innervation of the mouse and guinea pig corneal epithelium.

This study used immunohistochemistry, retrograde tracing, and high-resolution confocal microscopy to explore the structure and neurochemistry of nerve terminals in the corneal epithelium of mice and guinea pigs. In both species, sub-basal nerves formed a plexus in the basal epithelium. Some axons had bulbar endings within the basal epithelium, but most projected perpendicularly from sub-basal nerves to within a few micrometers of the epithelial surface. Three morphologies for these nerve terminals were identified. Simple terminals did not branch after leaving the sub-basal nerves and ended with a single, bulbar swelling. Ramifying terminals branched in the squamous cell layer, forming horizontal fibers that ran parallel to the surface and terminated with single bulbar swellings. Complex terminals branched as they approached the epithelial surface, forming a cluster of highly branched fibers with multiple bulbar endings. Calcitonin gene-related peptide immunolabeled (peptidergic) axons ended mostly in simple terminals, whereas transient receptor potential cation channel subfamily M member 8 immunolabeled (cold receptor) axons ended almost exclusively in complex terminals. Retrograde labeling identified discrete subpopulations of corneal afferent neurons in the trigeminal ganglion. Tyrosine hydroxylase-immunolabeled (sympathetic) nerve terminals originating from the superior cervical ganglion occurred throughout the corneal epithelium of mice, but only in the basal epithelium of guinea pigs. These findings demonstrate that nerve terminals in the corneal epithelium of mice and guinea pigs can be distinguished on the basis of their morphology and neurochemistry, and suggest that nerve terminals with different sensory modalities can be defined on the basis of their morphology.

Streptozotocin-induced diabetes differentially affects sympathetic innervation and control of plantar metatarsal and mesenteric arteries in the rat.

In humans neural control of arterial vessels supplying skin in the extremities is particularly vulnerable to the effects of diabetes. Here the streptozotocin (STZ) rat model of type 1 diabetes was used to compare effects on neurovascular function in plantar metatarsal arteries (PMAs), which supply blood to skin of hind paw digits, with those in mesenteric arteries (MAs). Twelve weeks after STZ (60 mg/kg ip), wire myography was used to assess vascular function. In PMAs, lumen dimensions were unchanged but both nerve-evoked contractions and sensitivity to α(1) (phenylephrine, methoxamine)- and α(2) (clonidine)-adrenoceptor agonists were reduced. The density of perivascular nerve fibers was also reduced by ~25%. These changes were not observed in PMAs from STZ-treated rats receiving either a low dose of insulin that did not greatly reduce blood glucose levels or a high dose of insulin that markedly reduced blood glucose levels. In MAs from STZ-treated rats, nerve-evoked increases in force did not differ from control but, because lumen dimensions were ~20% larger, nerve-evoked increases in effective transmural pressure were smaller. Increases in effective transmural pressure produced by phenylephrine or α,ß-methylene ATP in MAs from STZ-treated rats were not smaller than control, but the density of perivascular nerve fibers was reduced by ~10%. In MAs, the increase in vascular dimensions is primarily responsible for reducing effectiveness of nerve-evoked constrictions. By contrast, in PMAs decreases in both the density of perivascular nerve fibers and the reactivity of the vascular muscle appear to explain impairment of neurovascular transmission.

Hydrogen peroxide increases nerve-evoked contractions in mouse tail artery by an endothelium-dependent mechanism.

Reactive oxygen species contribute to regulating the excitability of vascular smooth muscle. This study investigated the actions of the relatively stable reactive oxygen species, H(2)O(2), on nerve-evoked contractions of mouse distal tail artery. H(2)O(2) (10-100 µM) increased nerve-evoked contractions of isometrically mounted segments of tail artery. Endothelium denudation increased nerve-evoked contractions and abolished the facilitatory effect of H(2)O(2). Inhibition of nitric oxide synthase with L-nitroarginine methyl ester (0.1mM) also increased nerve-evoked contractions and reduced the late phase of H(2)O(2)-induced facilitation. H(2)O(2)-induced facilitation of nerve-evoked contractions depended, in part, on synthesis of prostanoids and was reduced by the cyclooxygenase inhibitor indomethacin (1 µM) and the thromboxane A(2) receptor antagonist SQ 29548 (1 µM). H(2)O(2) increased sensitivity of nerve-evoked contractions to the α(2)-adrenoceptor antagonist idazoxan (0.1 µM) but not to the α(1)-adrenoceptor antagonist prazosin (10nM). Idazoxan and the α(2C)-adrenoceptor antagonist JP 1302 (0.5-1 µM) reduced H(2)O(2)-induced facilitation. H(2)O(2) induced facilitation of nerve-evoked contractions was abolished by the non-selective cation channel blocker SKF-96365 (10 µM), suggesting it depends on Ca(2+) influx. In conclusion, H(2)O(2)-induced increases in nerve-evoked contractions depended on an intact endothelium and were mediated by activating thromboxane A(2) receptors and by increasing the contribution of α(2)-adrenoceptors to these responses.

Sites of action of ghrelin receptor ligands in cardiovascular control.

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.