Perivascular pH Research Articles

Protons modulate perivascular axo-axonal neurotransmission in the rat mesenteric artery.

Previous studies have demonstrated that nicotine releases protons from adrenergic nerves via stimulation of nicotinic ACh receptors and activates transient receptor potential vanilloid-1 (TRPV1) receptors located on calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves, resulting in vasodilatation. The present study investigated whether perivascular nerves release protons, which modulate axon-axonal neurotransmission. Perfusion pressure and pH levels of perfusate in rat-perfused mesenteric vascular beds without endothelium were measured with a pressure transducer and a pH meter respectively. Periarterial nerve stimulation (PNS) initially induced vasoconstriction, which was followed by long-lasting vasodilatation and decreased pH levels in the perfusate. Cold-storage denervation of the preparation abolished the decreased pH and vascular responses to PNS. The adrenergic neuron blocker guanethidine inhibited PNS-induced vasoconstriction and effects on pH, but not PNS-induced vasodilatation. Capsaicin (CGRP depletor), capsazepine and ruthenium red (TRPV1 inhibitors) attenuated the PNS-induced decrease in pH and vasodilatation. In denuded preparations, ACh caused long-lasting vasodilatation and lowered pH; these effects were inhibited by capsaicin pretreatment and atropine, but not by guanethidine or mecamylamine. Capsaicin injection induced vasodilatation and a reduction in pH, which were abolished by ruthenium red. The use of a fluorescent pH indicator demonstrated that application of nicotine, ACh and capsaicin outside small mesenteric arteries reduced perivascular pH levels and these effects were abolished in a Ca(2+) -free medium. These results suggest that protons are released from perivascular adrenergic and CGRPergic nerves upon PNS and these protons modulate transmission in CGRPergic nerves.

Cerebral tissue oxygenation impairment during experimental cerebral malaria

Ischemia and hypoxia have been implicated in cerebral malaria (CM) pathogenesis, although direct measurements of hypoxia have not been conducted. C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) develop a neurological syndrome known as experimental cerebral malaria (ECM), whereas BALB/c mice are resistant to ECM. In this study, intravital microscopy methods were used to quantify hemodynamic changes, vascular/tissue oxygen (O2) tension (PO2), and perivascular pH in vivo in ECM and non-ECM models, employing a closed cranial window model. ECM mice on day 6 of infection showed marked decreases in pial blood flow, vascular (arteriolar, venular), and perivascular PO2, perivascular pH, and systemic hemoglobin levels. Changes were more dramatic in mice with late-stage ECM compared with mice with early-stage ECM. These changes led to drastic decreases in O2 delivery to the brain tissue. In addition, ECM animals required a greater PO2 gradient to extract the same amount of O2 compared with non-infected animals, as the pial tissues extract O2 from the steepest portion of the blood O2 equilibrium curve. ECM animals also showed increased leukocyte adherence in postcapillary venules, and the intensity of adhesion was inversely correlated with blood flow and O2 extraction. PbA-infected BALB/c mice displayed no neurological signs on day 6 and while they did show changes similar to those observed in C57BL/6 mice (decreased pial blood flow, vascular/tissue PO2, perivascular pH, hemoglobin levels), non-ECM animals preserved superior perfusion and oxygenation compared with ECM animals at similar anemia and parasitemia levels, resulting in better O2 delivery and O2 extraction by the brain tissue. In conclusion, direct quantitative assessment of pial hemodynamics and oxygenation in vivo revealed that ECM is associated with severe progressive brain tissue hypoxia and acidosis.

Paracrine control of mesenteric perivascular axo-axonal interaction

Immunohistochemical study of rat mesenteric arteries showed dense innervation of adrenergic nerves, calcitonin gene-related peptide (CGRP)-containing nerves (CGRPergic nerves), nitric oxide-containing nerves (nitrergic nerves). Double-immunostaining revealed that most CGRPergic or nitrergic nerves were in close contact with adrenergic nerves. CGRPergic and transient receptor potential vanilloid-1 (TRPV1)-immunopositive nerves appeared in the same neurone. In rat perfused mesenteric vascular beds without endothelium and with active tone, perfusion of nicotine, or bolus injection of capsaicin and acetylcholine and periarterial nerve stimulation (PNS) lowered pH levels of out flowed perfusate concomitant with vasodilation. Cold-storage denervation of preparations abolished pH lowering induced by nicotine and PNS. Guanethidine inhibited PNS- and nicotine-, but not acetylcholine- and capsaicin-, induced pH lowering. Pharmacological analysis showed that protons were released not only from adrenergic nerves but also from CGRPergic nerves. A study using a fluorescent pH indicator demonstrated that nicotine, acetylcholine and capsaicin applied outside small mesenteric artery lowered perivascular pH levels, which were not observed in Ca(2+) free medium. Exogenously injected hydrochloric acid in denuded preparations induced pH lowering and vasodilation, which was inhibited by denervation, TRPV1 antagonists and capsaicin without affecting pH lowering. These results suggest that excitement of adrenergic nerves releases protons to activate TRPV1 in CGRPergic nerves and thereby induce vasodilation. It is also suggested that CGRPergic nerves release protons with exocytosis to facilitate neurotransmission via a positive feedback mechanism.

Augmentation of NO-mediated vasodilation in metabolic acidosis

Reduction of perivascular pH in acidemia produces hyporesponsiveness of vascular bed to vasoconstrictors. In the present study, we examined the effects of modest acidification on dilatory responses of isolated rat thoracic aorta. Acetylcholine produced endothelium-dependent relaxation in phenylephrine-precontracted aorta, which was markedly enhanced by acidification of Krebs-Henseleit solution from pH 7.4 to 7.0. A similar augmentation was observed in the relaxing responses to NO donors (SNP, SIN-1, SNAP), 8-Br-cGMP and NS-1619 (a putative K Ca channel opener and/or Ca channel inhibitor) in endothelium-denuded, phenylephrine-contracted aorta. However, papaverine-induced relaxation was not affected by the change in pH. At pH 7.4, the relaxing responses to acetylcholine and SNP were partially inhibited by charybdotoxin (K Ca channel inhibitor) but not glibenclamide (K ATP channel inhibitor), while at pH 7.0 the relaxation induced by either drug was not affected by K + channel inhibitors. Relaxation induced by 8-Br-cGMP or NS-1619 was not inhibited by charybdotoxin or glibenclamide. Acidification to pH 7.0 increased the cGMP production in response to acetylcholine in endothelium-intact aorta and to SNP in endothelium-denuded aorta. These results show that modest acidification augments NO-mediated relaxation in rat aorta, probably due to an enhancement of cGMP-dependent but K + channel-unrelated relaxation mechanisms.

A mathematical model of cerebral blood flow chemical regulation--Part I: Diffusion processes.

This paper proposes a mathematical model which describes the production and diffusion of vasoactive chemical factors involved in oxygen-dependent cerebral blood flow (CBF) regulation in the rat. Partial differential equations describing the relations between input and output variables have been replaced with simpler ordinary differential equations by using mathematical approximations of the hyperbolic functions in the Laplace transform domain. This model is composed of two submodels. In the first, oxygen transport from capillary blood to cerebral tissue is analyzed to link changes in mean tissue oxygen pressure with CBF and arterial oxygen concentration changes. The second submodel presents equations describing the production of vasoactive metabolites by cerebral parenchyma, due to a lack of oxygen, and their diffusion towards pial perivascular space. These equations have been used to simulate the time dynamics of mean tissue PO2, perivascular adenosine concentration, and perivascular pH to changes in CBF. The present simulation points out that the time delay introduced by diffusion processes is negligible if compared with the other time constants of the system under study. In a subsequent work the same equations will be included in a model of the cerebral vascular bed to clarify the metabolite role in CBF regulation.

Blood-brain barrier to hydrogen ion during acute metabolic acidosis in piglets.

The present study investigates the integrity of the blood-brain barrier to H+ or HCO3- during acute plasma acidosis in 35 newborn piglets anesthetized with pentobarbital sodium. Cerebrospinal fluid acid-base balance, cerebral blood flow (CBF), and cerebral oxygenation were measured after infusion of HCl (0.6 N, 0.191-0.388 ml/min) for a period of 1 h at a constant arterial PCO2 of 35-40 Torr. HCl infusion resulted in decreased arterial pH from 7.38 +/- 0.01 to 7.00 +/- 0.02 (P less than 0.01). CBF measured by the tracer microsphere technique was decreased by 12% from 69 +/- 6 to 61 +/- 4 ml.min-1.100 g-1 (P less than 0.05). Infusion of 0.6 N NaCl as a hypertonic control had no effect on CBF. Cerebral metabolic rate for O2 and O2 extraction was not significantly changed from control (3.83 +/- 0.20 ml.min-1.100 g-1 and 5.7 +/- 0.6 ml/100 ml, respectively) during acid infusion. Cerebral venous PO2 was increased from 41.6 +/- 2.1 to 53.8 +/- 4.0 Torr by HCl infusion (P less than 0.02) associated with a shift in O2-hemoglobin affinity of blood in vivo from 38 +/- 2 to 50 +/- 1 Torr. Cisternal cerebrospinal fluid pH decreased from 7.336 +/- 0.014 to 7.226 +/- 0.027 (P less than 0.005), but cerebrospinal fluid HCO3- concentration was not changed from control (25.4 +/- 1.0 meq/l). These data suggest that there is a functional blood-brain barrier in newborn piglets, that is relatively impermeable to HCO3- or H+ and maintains cerebral perivascular pH constant in the face of acute severe arterial acidosis. (ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine response on pial arteries, influence of CO2 and blood pressure.

The television image-splitting technique was used to study the influence of arterial pCO2 and blood pressure on the dilatatory response of pial arterioles to topically applied adenosine in chloralose anaesthetised cats. At normocapnia (pCO2 congruent to 35 mm Hg) 10(-5) adenosine caused pial arteriole dilatation of 29.2 +/- 2.7% (S.E.M.). This was significantly reduced to 14.5 +/- 1.6% (P < 0.001) at pCO2 25 mm Hg and to 8.5 +/- 1.6% (P Ø 0.001) at pCO2 48 mm Hg. Lowering the blood pressure to 65--85 mm Hg had no significant effect on the adenosine response, but raising the blood pressure to 140--160 mm Hg significantly reduced the adenosine response to 22.1 +/- 1.8% (P < 0.005). The response was independent of vessel size except at hypertension where vessels < 150 micrometer were significantly more reactive than the larger vessels (P < 0.01). These results indicate that adenosine induced vasodilatation of pial arterioles shows little change in the face of alterations in vessel tone induced by altering blood pressure, but is markedly decreased by the combination of changing perivascular pH and vascular resistance through moderate changes in arterial pCO2. The importance of these results in assessing the role of adenosine as a cerebral vasodilator is discussed.

Unimportance of perivascular H+ AND K+ activities for the adjustment of pial arterial diameter during changes of arterial blood pressure in cats.

The role of perivascular H+ and K+ in the adjustment of pial arterial diameter during changes in arterial blood pressure was investigated in chloralose anesthetized cats. Blood pressure was reduced by i.v. mecamylamine or pentolinium and was increased by i.v. hypertensin. Pial arterioles and arteries with a control diameter ranging from 37--218 microns at a spontaneous mean arterial blood pressure of 128 +/- 16 (SD) mm Hg were studied. Vascular diameter as measured by TV image splitting showed the typical reactions, i.e. constriction during increase (up to 200 mm Hg) and dilation during decrease in blood pressure (down to 60 mm Hg). Perivascular H+ and K+ activities were measured using pH microelectrodes (Hinke type) and K+ ion exchanger microelectrodes, respectively. Under control conditions perivascular pH was 7.25 +/- 0.11 (SD) and K+ activity was 2.46 +/- 0.65 (SD) mM, respectively. During changes in blood pressure the vascular reactions of pial arteries were not accompanied by significant alterations in perivascular H+ or K+ activity. From these data it can be concluded that mechanisms other than those which are mediated by H+ or K+ are involved in the adjustment of pial arterial diameter during changes in arterial blood pressure.

The use of microelectrodes for measurement of local H+ activity in the cortical subarachnoidal space of cats.

pH microelectrodes with pointed tip (Hinke-type) were constructed for the continuous measurement of the local pH in the perivascular space of pial arteries in the feline cerebral cortex. The sensitive tip had a length of 20-60 mu and a base diameter of 10-25 mu. As reference electrode, a micropipette (tip diameter 2 mu), filled with 150 mM KCl was used. Calibration curves were linear and showed a sensitivity of 54.5-57.5 mV/pH unit at 38 degrees C. Advantages of such electrodes are the easy penetration of the subarachnoid membrane, the long life span, the quick response, and a minimal drift. The electrodes were tested in vivo during hyper- and hypoventilation and during local perivascular injection of mock spinal fluid at varying pH. A close correlation was observed between the change in perivascular pH and the corresponding change in pial arterial diameter.

Influence of pH and pCO2 on alpha-receptor mediated contraction in brain vessels.

The response of isolated brain vessels to various pH levels or carbon dioxide tensions was analyzed. Reduction of the pH induced a slight relaxation of the vessel, whereas an increase in the pH produced a slight contraction. These effects were markedly exaggerated when the alpha-adrenergic receptors in the vascular wall were activated by noradrenaline. During these conditions the contractile response to noradrenaline was reduced by about 40% at a pH of 7.01, while, on the other hand, the response was enhanced 3-fold at a pH of 7.80. Variations in carbon dioxide tension of the buffer solution between 16 mmHg and 64 mmHg produced no consistent change, provided the pH remained constant. The results indicate that an interaction between the perivascular pH and the adrenergic alpha-receptor mediated contraction in brain vessels may occur.

Hyperventilation and cerebral blood flow.

Hypocapnic-hyperventilation has a profound, but probably temporary, effect on CBF, producing approximately a 2% decline in CBF for each 1 torr decline in P co co2 . This effect appears to be mediated through changes in perivascular pH of the cerebral resistance vessels acting directly on the vessel wall. At low P co co2 the vasoconstrictor effect of short-term hypocapnic-hyperventilation is attenuated by resultant cerebral hypoxia. During prolonged hyperventilation CBF returns toward normal as the pH in the CSF is restored. Short-term hypocapnic-hyperventilation can be lifesaving in the treatment of acute intracranial hypertension. On the other hand, prolonged hyperventilation has not been convincingly shown to benefit patients, whether with severe head injury or cerebral infarction, or during carotid endarterectomy without bypass.

Micropuncture evaluation of the importance of perivascular pH for the arteriolar diameter on the brain surface.

A micropipette technique was used to induce local changes of the bicarbonate concentration of the cerebro-spinal fluid surrounding arterioles on the exposed cerebral cortex of anaesthetized rats and cats. Injection volumes of a few nanoliters caused circumscribed and pronounced changes of the diameter of the arterioles under study: mock spinal fluid without bicarbonate dilated, while a solution containing 25 meq/l of bicarbonate constricted the vessels. In such experiments the localpCO2 of the arteriolar wall remains practically constant, since it is set by thepCO2 of the arterial blood and of the cerebral tissue. Hence the microinjections essentially consisted in a local change of the pH of the fluid surrounding a small segment of a cerebral arteriole. Since metabolic changes of the nervous tissue changes the periarteriolar pH, it is probable that local pH induced vasomotor changes of the type reported here participate in the so called metabolic regulation of the cerebral blood flow which underlies the local adaptation of the cerebral blood flow to changing functional demands.