Lysophosphatidic acid induces shear stress-dependent contraction in mouse aortic strip in situ.

We previously reported that lysophosphatidic acid (LPA) regulates Ca²âº influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²âº responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²âº responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²âº oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A2 receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²âº-dependent contraction in medial smooth muscle cells. Thromboxane A2 may be involved in that response.

Parasympathetic reflex vasodilatation in the masseter muscle compensates for carotid hypoperfusion during the vagus-mediated depressor response.

Parasympathetic vasodilatation in the orofacial area is thought to be an important factor in the regulation of blood flow in the common carotid artery (CABF), and disturbances in parasympathetic vasodilatations may be related to impairment of the CABF inducing craniofacial ischemia. We hypothesized that the parasympathetic vasodilatation in the masseter muscle evoked by a vagus-mediated reflex is involved in the maintenance of the CABF during the vagus-mediated depressor response. In the present study, we compared changes in blood flow in the masseter muscle (MBF) and CABF, and systemic arterial blood pressure (SABP) evoked by electrical stimulation of the central cut end of the cervical vagus nerve (cVN) in anesthetized and sympathectomized rats. Electrical stimulation of the cVN in the sympathectomized animals caused an increase in MBF followed by a CABF increase, although it simultaneously induced a decrease in SABP. These increases in blood flow changed to decreases after intravenous administration of atropine (100 µg/kg), while pretreatment with atropine had no effect on the changes in SABP. Microinjection (50 nl/site) of the muscimol (1mM), into the nucleus of the solitary tract, which is involved in reflex cardiovascular regulation, markedly inhibited the cVN stimulation-induced MBF increase. Our results indicate that vagal-parasympathetic vasodilatation in the masseter muscle compensates for carotid hypoperfusion during the vagus-mediated depressor response, and that GABAergic neurons may be involved in the inhibition of this response. This inhibition may result in the impairment of CABF, suggesting an important role in the etiology of neurally mediated syncope.

Vagal visceral inputs to the nucleus of the solitary tract: involvement in a parasympathetic reflex vasodilator pathway in the rat masseter muscle.

The present study examined whether vagal visceral inputs are involved in parasympathetic reflex vasodilatation in the masseter muscle in urethane-anesthetized and cervically sympathectomized rats. Electrical stimulation of the central cut end of the cervical vagus nerve (VN) including visceral afferent fibers, which consisted of cervical/thoracic branches (heart and lungs) and abdominal branches (entire gastrointestinal tract), elicited intensity- and frequency-dependent increases of blood flow in the masseter muscle (MBF). Activation of the abdominal VN inferior to the diaphragm failed to affect the MBF. MBF increases evoked by cervical VN stimulation were reduced significantly by hexamethonium. Pretreatment with atropine reduced the MBF increase evoked by VN stimulation significantly, whereas pretreatment with either propranolol or phentolamine had no effect on the response. MBF increases occurred with electrical stimulation of nucleus of the solitary tract (NTS), and these increases were significantly reduced by the administration of hexamethonium and atropine. MBF increases also occurred after microinjection of glutamate into the NTS in a dose-dependent manner. Microinjection of muscimol into the NTS caused a significant attenuation of the VN stimulation-induced MBF increases. Our results suggest that vagal visceral inputs passing to the NTS are involved in the parasympathetic reflex vasodilatation in the rat masseter muscle. The MBF increase evoked by the vagal-parasympathetic reflex mechanism occurred via visceral afferents running in the cervical VN, but not in the abdominal VN, suggesting that the vagal visceral afferents derived from cardiovascular and/or respiratory systems may play an important role in the regulation of the MBF.

Regional differences in blood flow variation in rat masseter muscle.

Regions of a belly in a masseter muscle have been suggested to be activated independently in order to enable complex jaw-movements. However a regional difference of the masseter blood flow (MBF) is still unclear although the blood flow is one of the most important factors during activation of the muscle. The present study examined regional differences in blood flow in rat masseter muscle by comparing blood flow values at the inferior, centre, superior, anterior, and posterior regions of the muscle belly using a laser speckle imaging flowmeter with or without sympathetic and parasympathetic stimulation. Regional differences in blood flow levels were observed in each region of the masseter muscle belly during rest. Additionally, amplitudes of blood flow changes evoked by electrical stimulation of parasympathetic and sympathetic nerves differed among regions. These results demonstrate the regional differences in hemodynamics during rest, sympathetic vasoconstriction (including the recovery phase), and parasympathetic vasodilatation in rat masseter muscle.

Parasympathetic vasodilator fibers in rat digastric muscle.

The present study examined whether parasympathetic vasodilator fibers exist in rat jaw-opening muscles such as the digastric muscle. The mental nerve was stimulated to activate the parasympathetic vasodilator nerve in the digastric muscle. Electrical stimulation of the mental nerve elicited intensity- and frequency-dependent increases of blood flow in this muscle. These increases were markedly reduced by hexamethonium in dose- and time-dependent manners, but pretreatment with phentolamine or propranolol had no effect. Pretreatment with atropine also attenuated the increase in blood flow in digastric muscle. When retrograde fluorogold was injected into the digastric muscle, labeled neurons were observed in the otic ganglion only on the ipsilateral side, but not in the pterygopalatine ganglion of either side. These results indicate that parasympathetic vasodilator fibers originate from cell bodies in the otic ganglion in rat digastric muscle.

Involvement of vasoactive intestinal polypeptide in the parasympathetic vasodilatation of the rat masseter muscle.

The parasympathetic vasodilatory fibres are known to innervate vessels in a rat masseter muscle via both cholinergic and non-cholinergic mechanisms. However, the non-cholinergic mechanisms are still unclear. Recently, vasoactive intestinal polypeptide (VIP) was convincingly shown to be involved in the parasympathetic vasodilatation in orofacial areas, such as submandibular glands and lower lip. However, very little is known about the rat masseter muscle. The present study was designed in the rat masseter muscle to assess (1) whether the parasympathetic nerve innervating vessels have VIP immunoreactivities, (2) whether intravenous administration of VIP induces the vasodilatation, and (3) effects of selective VIP receptor antagonist ([4Cl-d-Phe(6), Leu(17)] VIP) in the presence or absence of atropine on the parasympathetic vasodilatation. The VIP immunoreactivities were found at two sites of the parasympathetic otic ganglion and nerve fibres located around vessels. The intravenous administration of VIP induced the vasodilatation, and [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the vasodilatation evoked by VIP administration. The parasympathetic vasodilatation was not inhibited by [4Cl-d-Phe(6), Leu(17)] VIP. However, treatment with [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the parasympathetic vasodilatation when [4Cl-d-Phe(6), Leu(17)] VIP was administered together with atropine. These results suggest that (1) VIP exists in the postganglionic parasympathetic nerve innervating the vessels in the masseter muscle, (2) the intravenous administration of VIP induces the vasodilatation in the masseter muscle, and (3) VIP may be involved in the parasympathetic vasodilatation in the masseter muscle when muscarinic cholinergic receptors are deactivated by either atropine or the suppression of the ACh release.

Difference between male and female rats in cholinergic activity of parasympathetic vasodilatation in the masseter muscle.

We compared the changes in blood flow of the masseter muscle (MBF), lower lip (LBF) and common carotid artery (CCABF) evoked by electrical stimulation of the lingual nerve (LN) in order to examine whether high cholinergic activity of parasympathetic vasodilatation in females is specific for the masseter muscle, and whether sex-associated differences in cholinergic parasympathetic vasodilatation affect the regulation of blood flow to the orofacial area from the CCABF in urethane-anaesthetized, vago-sympathectomized male and female rats. Increases in the MBF, LBF and CCABF evoked by LN stimulation appear to be mediated via an activation of parasympathetic reflex vasodilatation since these increases were profoundly reduced by pretreatment with the autonomic cholinergic ganglion blocker hexamethonium (10 mg/kg). Although alpha- and beta-adrenoceptor antagonists (phentolamine and propranolol, 100 microg/kg) had no effect on the LN stimulation-induced blood flow increases in either sex, a marked difference was found between males and females in the effects of the antimuscarinic agent atropine (1-100 microg/kg) on these blood flow increases. Pretreatment with atropine slightly attenuated the increase in the MBF in males, but in females it markedly reduced the increases in all three sites measured, especially in the MBF. Our results suggest that (1) cholinergic activity of the parasympathetic vasodilatation in females is higher than that in males in most orofacial tissues, but particularly in the masseter muscle and (2) cholinergic parasympathetic vasodilatations are more involved in the regulation of blood flow to the orofacial area from the CCABF in females than in males.

Circulating adrenaline released by sympathoadrenal activation elicits acute vasodilatation in the rat masseter muscle.

The present study was designed to examine the effects of circulating catecholamines released by sympathoadrenal system on the haemodynamics of the masseter muscle in deeply urethane-anaesthetized, artificially ventilated, cervically vagotomized and sympathectomized rats. Intravenous administration of adrenaline induced a biphasic change of blood flow in the masseter muscle (MBF). The change of blood flow showed an initial marked increase and successive slight decrease in a dose-dependent manner (0.01-1 microg/kg). The administration of noradrenaline had no significant effect on the MBF. The increase in the MBF evoked by exogenously applied adrenaline was markedly reduced by the intravenous administration of propranolol (100 microg/kg), whereas pretreatment with either hexamethonium (10 mg/kg), atropine (100 microg/kg), or phentolamine (1 mg/kg) failed to affect the MBF increase. Electrical stimulation of splanchnic nerve (SPLN) preganglionic neurones projecting to the adrenal medulla elicited frequency-dependent (1-20 Hz) increases in the MBF. The intravenous administration of the beta(2)-adrenergic receptor selective antagonist, ICI 118551 (0.5 mg/kg), almost abolished the MBF increase induced by SPLN stimulation, but pretreatment with the beta(1)-adrenergic receptor selective antagonist, atenolol (1 mg/kg), had no effect on this response. The results of the present study indicate that circulating adrenaline elicits acute vasodilatation through a beta-adrenergic mechanism in the rat masseter muscle. Vascular beta(2)-adrenergic receptors in the masseter muscle may be activated preferentially by adrenaline released from the adrenal medulla, suggesting that the sympathoadrenal system is involved in the marked MBF increase during sympathoexcitation.

Inhibitory effects of excess sympathetic activity on parasympathetic vasodilation in the rat masseter muscle.

The present study was designed to examine the effect of sympathetic tonic activity on parasympathetic vasodilation evoked by the trigeminal-mediated reflex in the masseter muscle in urethane-anesthetized rats. Sectioning of the superior cervical sympathetic trunk (CST) ipsilaterally increased the basal level of blood flow in the masseter muscle (MBF). Electrical stimulation of the peripheral cut end of the CST for 2 min using 2-ms pulses ipsilaterally decreased in a dependent manner the intensity (0.5-10 V) and frequency (0.1-5 Hz) of the MBF. The CST stimulation for 2 min at <0.5 Hz with 5 V using 2-ms pulses seems to be comparable with the spontaneous activity in the CST fibers innervating the masseter vasculature, because this stimulation restored the basal level of the MBF to the presectioned values. Parasympathetic vasodilation evoked by electrical stimulation of the central cut end of the lingual nerve in the masseter muscle was markedly reduced by CST stimulation for 2 min with 5 V using 2-ms pulses in a frequency-dependent manner (0.5-5 Hz). Intravenous administration of phentolamine significantly reduced the vasoconstriction induced by CST stimulation in a dose-dependent manner (0.1-1 mg/kg), but pretreatment with either phentolamine or propranolol failed to affect the sympathetic inhibition of the parasympathetic vasodilation. Our results suggest that 1) excess sympathetic activity inhibits parasympathetic vasodilation in the masseter muscle, and 2) alpha- and beta-adrenoceptors do not contribute to sympathetic inhibition of parasympathetic vasodilation, and thus some other types of receptors must be involved in this response.

Evidence for parasympathetic vasodilator fibres in the rat masseter muscle.

The present study was designed to examine (1) whether there are vasodilator fibres in the masseter muscle, and (2) if there are, to establish the neural pathways mediating these responses in urethane-anaesthetized rats. Electrical stimulation of the central cut end of the lingual nerve (LN) elicited intensity- and frequency-dependent increases of the blood flow in the masseter muscle (MBF) and lower lip (LBF). Increases in both the MBF and LBF evoked by the LN stimulation were reduced by hexamethonium in a dose-dependent manner (1-10 mg kg(-1)). Pretreatment with phentolamine or propranolol at a dose of 100 microg kg(-1) had no effect on the increases in either MBF or LBF evoked by LN stimulation. Pretreatment with atropine (100 microg kg(-1)) significantly reduced the MBF increase induced by LN stimulation, but not that in the LBF. The sectioning of the superior cervical sympathetic trunk did not affect the responses. MBF increases occurred with electrical stimulation of the trigeminal ganglion, and these increases were significantly reduced by the administration of hexamethonium and atropine. Lidocaine microinjection into the trigeminal spinal nucleus or salivatory nuclei caused a significant attenuation of the LN-induced MBF increases. When wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was injected into the masseter muscle, labelled neurones were abundantly observed in the otic ganglion. The present study indicates that there are parasympathetic cholinergic and noncholinergic vasodilator fibres originating from cell bodies in the otic ganglion in the rat masseter muscle. The MBF increase evoked by activation of the parasympathetic fibres occurred via the trigeminal mediated reflex, suggesting that the novel parasympathetic vasodilator response may play an important role in the regulation of the haemodynamics of jaw muscles.

[Role of lysophosphatidic acid as a mechanosensitizer].

The mechanotransduction mechanisms play an important role in regulation of specific cellular response or maintenance of cellular homeostasis in a wide variety of cell types. Increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is an important signal in the first step of mechanotransduction. Mechanosensitive (MS) cation channels are thought to be a putative pathway of Ca(2+) entry; however, the molecular mechanisms remain unclear. We have previously demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid present in human plasma, sensitizes the response of [Ca(2+)](i) to mechanical stress in cultured smooth muscle cells, cultured lung epithelial cells, and cultured lens epithelial cells. Using real-time confocal microscopy, local increases in [Ca(2+)](i) in several regions within the cell subjected to mechanical stress were clearly visualized in cultured bovine lens epithelial cells and cultured vascular endothelial cells in the presence of LPA. We called the phenomenon "Ca(2+) spots". Pharmacological studies revealed that the Ca(2+) spot is an elementary Ca(2+)-influx event through MS channels. In this review, possible physiological and pathophysiological roles of LPA as a mechanosensitizer are discussed.

Optical bioimaging: from living tissue to a single molecule: calcium imaging in blood vessel in situ employing two-photon excitation fluorescence microscopy.

Recent developments in optoelectronics permit real-time Ca(2+) imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca(2+) imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-microm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 microm). When serial sectioning of 21 sequential images at 0.3-microm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca(2+)-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca(2+)](i) of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca(2+) imaging in blood vessels in situ.