Wojciech A. Kosiba

Cutaneous Active Vasodilation in Humans Is Mediated by Cholinergic Nerve Cotransmission

During heat stress, increases in blood flow in nonglabrous skin in humans are mediated through active vasodilation by an unknown neurotransmitter mechanism. To investigate this mechanism, a three-part study was performed to determine the following: (1) Is muscarinic receptor activation necessary for active cutaneous vasodilation? We iontophoretically applied atropine to a small area of forearm skin. At that site and an untreated control site, we measured the vasomotor (laser-Doppler blood flow [LDF]) and sudomotor (relative humidity) responses to whole-body heat stress. Blood pressure was monitored. Cutaneous vascular conductance (CVC) was calculated (LDF divided by mean arterial pressure). Sweating was blocked at treated sites only. CVC rose at both sites (P < .05 at each site); thus, cutaneous active vasodilation is not effected through muscarinic receptors. (2) Are nonmuscarinic cholinergic receptors present on cutaneous arterioles? Acetylcholine (ACh) was iontophoretically applied to forearm skin at sites pretreated by atropine iontophoresis and at untreated sites. ACh increased CVC at untreated sites (P < .05) but not at atropinized sites. Thus, the only functional cholinergic receptors on cutaneous vessels are muscarinic. (3) Does cutaneous active vasodilation involve cholinergic nerve cotransmission? Botulinum toxin was injected intradermally in the forearm to block release of ACh and any coreleased neurotransmitters. Heat stress was performed as in part 1 of the study. At treated sites, CVC and relative humidity remained at baseline levels during heat stress (P > .05). Active vasodilator and sudomotor responses to heat stress were abolished by botulinum toxin. We conclude that cholinergic nerve activation mediates cutaneous active vasodilation through release of an unknown cotransmitter, not through ACh.

Baroreflex control of the cutaneous active vasodilator system in humans.

Cutaneous arterioles are controlled by vasoconstrictor and active vasodilator sympathetic nerves. To find out whether the active vasodilator system is under baroreceptor control, laser-Doppler velocimetry and the local iontophoresis of bretylium were combined to allow selective study of the active vasodilator system. Each of six subjects had two forearm sites (0.64 cm2) treated with bretylium to abolish adrenergic vasoconstrictor control. Laser-Doppler velocimetry was monitored at those sites and at two adjacent untreated sites. Subjects underwent 3 minutes of lower-body negative pressure (LBNP) and 3 minutes of cold stress to verify blockade of vasoconstrictor nerves. They were then subjected to whole-body heat stress (water-perfused suits), and the 3 minutes of LBNP was repeated. Finally, subjects were returned to normothermia, and LBNP and cold stress were repeated to verify the persistence of blockade. During the application of LBNP in normothermia, cutaneous vascular conductance (CVC) fell at untreated sites by 22.7 +/- 4.7% (p less than 0.01) but was unaffected at bretylium-treated sites (p greater than 0.20). During cold stress, CVC at untreated sites fell by 30.2 +/- 1.7% (p less than 0.01) and at treated sites rose by 0.7 +/- 4.6% (p greater than 0.10). Both control and bretylium-treated sites reflexly vasodilated in response to hyperthermia. With LBNP during hyperthermia, CVC at untreated sites fell by 23.3 +/- 7.1% (p less than 0.05) and at treated sites 17.9 +/- 9.2% (p less than 0.05) with no significant difference between sites (p greater than 0.10). After return to normothermia, neither LBNP application nor cold stress caused CVC to fall at treated sites (p greater than 0.10). Thus, the vasoconstrictor system was blocked by bretylium treatment throughtout the study, whereas the active vasodilator response to heat stress was intact. Because LBNP in hyperthermia induced similar falls in CVC at both sites, we conclude that baroreceptor unloading elicits a withdrawal of active vasodilator tone and that the baroreflex has control of the active vasodilator system.

The involvement of nitric oxide in the cutaneous vasoconstrictor response to local cooling in humans

Cutaneous vascular conductance (CVC) declines in response to local cooling (LC). Previous work indicates that at least part of the vasoconstrictor response to LC may be through an inhibitory effect on nitric oxide synthase (NOS) activity. In this study we further tested that notion. A total of eight (6 male, 2 female) subjects participated (Part 1 n= 7; Part 2 n= 5, 4 of whom participated in Part 1). Skin blood flow was monitored by laser‐Doppler flowmetry. Control of local skin and body temperatures was achieved with Peltier cooler/heater probe holders and water perfused suits, respectively. Microdialysis fibres were inserted aseptically. Saline, l‐NAME (20 mm; to inhibit NOS activity) and sodium nitroprusside (SNP 10 μm) were infused by microdialysis. Bretylium tosylate (BT), to block adrenergic function, was administered by iontophoresis. CVC was calculated from blood flow and blood pressure. Part 1 was designed to determine the relative roles of the NO and the adrenergic systems. The infusion of l‐NAME elicited a 35 ± 4% decrease in CVC at the l‐NAME and BT +l‐NAME sites (P < 0.05); subsequent slow LC (34–24°C) for 35 min caused a significant (P < 0.05) decrease in CVC at control sites (68 ± 4%) and at the BT treated sites (39 ± 5%). LC caused a further 23 ± 5% of initial baseline decrease in CVC at the l‐NAME treated sites (P < 0.05). Importantly, CVC at the BT +l‐NAME sites was unaffected by LC (P > 0.05). Part 2 was designed to test whether LC influences were specific to the NOS enzymes. Two sites were pretreated with both BT and l‐NAME. After 50 min, SNP was added as an NO donor to restore baseline CVC at one site. The same LC process as in Part 1 was applied. There was a 24 ± 10% decrease (P < 0.05) in CVC at sites with baseline CVC restored, while, as in Part 1, there was no change (P > 0.05) at sites treated with BT +l‐NAME only. These data suggest that the vasoconstriction with slow LC is due to a combination of increased noradrenaline release and decreased activity of both NOS per se and of process(es) downstream of NOS.

The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans

Presynaptic blockade of cutaneous vasoconstrictor nerves (VCN) abolishes the axon reflex (AR) during slow local heating (SLH) and reduces the vasodilator response. In a two-part study, forearm sites were instrumented with microdialysis fibers, local heaters, and laser-Doppler flow probes. Sites were locally heated from 33 to 40 degrees C over 70 min. In part 1, we tested whether this effect of VCN acted via nitric oxide synthase (NOS). In five subjects, treatments were as follows: 1) untreated; 2) bretylium, preventing neurotransmitter release; 3) N(G)-nitro-L-arginine methyl ester (L-NAME) to inhibit NOS; and 4) combined bretylium + L-NAME. At treated sites, the AR was absent, and there was an attenuation of the ultimate vasodilation (P < 0.05), which was not different among those sites (P > 0.05). In part 2, we tested whether norepinephrine and/or neuropeptide Y is involved in the cutaneous vasodilator response to SLH. In seven subjects, treatments were as follows: 1) untreated; 2) propranolol and yohimbine to antagonize alpha- and beta-receptors; 3) BIBP-3226 to antagonize Y(1) receptors; and 4) combined propranolol + yohimbine + BIBP-3226. Treatment with propranolol + yohimbine or BIBP-3226 significantly increased the temperature at which AR occurred (n = 4) or abolished it (n = 3). The combination treatment consistently eliminated it. Importantly, ultimate vasodilation with SLH at the treated sites was significantly (P < 0.05) less than at the control. These data suggest that norepinephrine and neuropeptide Y are important in the initiation of the AR and for achieving a complete vasodilator response. Since VCN and NOS blockade in combination do not have an inhibition greater than either alone, these data suggest that VCN promote heat-induced vasodilation via a nitric oxide-dependent mechanism.

The effect of microdialysis needle trauma on cutaneous vascular responses in humans

Microdialysis enables in-depth mechanistic study of the cutaneous circulation in humans. However, whether the insertion or presence of the microdialysis fiber (MDF) affects the skin circulation or its responses is unknown. We tested whether the cutaneous vascular response to whole body heating (WBH) was affected by MDF or by pretreatment with ice (part 1) or local anesthesia (LA; part 2). Eleven subjects participated, 9 in part 1 and 8 in part 2 (5 participated in both). In both parts, four sites on the forearm were selected, providing untreated control, MDF only, ice or LA only, and combined MDF plus ice or LA. A tube-lined suit controlled whole body skin temperature, which was raised to approximately 38 degrees C for WBH. Skin sites were instrumented with laser-Doppler flow probes. Data were expressed as cutaneous vascular conductance (CVC). Baseline levels were not different among sites (P > 0.05). In part 1, the internal temperature for the onset of vasodilation was higher (P > 0.05) with MDF with or without ice pretreatment than at untreated control sites (control 36.6 +/- 0.1 degrees C, Ice 36.5 +/- 0.1, MDF 36.8 +/- 0.1 degrees C, and Ice+MDF 36.8 +/- 0.1 degrees C). Peak CVC during WBH was decreased (P < 0.05) by MDF (control 73 +/- 7 vs. MDF 59 +/- 6% of maximal CVC). Ice (73 +/- 6% of maximal CVC) or Ice+MDF (69 +/- 6% of maximal CVC) did not affect (P > 0.05) peak CVC compared with control. In part 2, the temperature threshold for the onset of vasodilation was increased by MDF with or without LA treatment and by LA alone (P < 0.05; control 36.6 +/- 0.1 degrees C, MDF 36.7 +/- 0.1 degrees C, LA 36.8 +/- 0.1 degrees C, and LA+MDF 36.8 +/- 0.1 degrees C). Peak CVC was decreased by MDF (control 69 +/- 6% of maximal CVC vs. MDF 58 +/- 8% of maximal CVC; P < 0.05). LA only (65 +/- 10% of maximal CVC) or MDF in the presence of LA (73 +/- 12% of maximal CVC) did not affect (P > 0.05) peak CVC compared with control. Thus LA or MDF increases the temperature threshold for the onset of vasodilation. MDF alone decreases the peak vasodilator response in CVC to WBH; however, this attenuation did not occur if ice or LA is used before MDF placement. Ice or LA alone do not affect the peak response in CVC to WBH. How those treatments prevent or reverse the effect of MDF placement is presently unclear.

Exogenous melatonin administration modifies cutaneous vasoconstrictor response to whole body skin cooling in humans

Abstract:  Humans and other diurnal species experience a fall in internal temperature (Tint) at night, accompanied by increased melatonin and altered thermoregulatory control of skin blood flow (SkBF). Also, exogenous melatonin induces a fall in Tint, an increase in distal skin temperatures and altered control of the cutaneous active vasodilator system, suggesting an effect of melatonin on the control of SkBF. To test whether exogenous melatonin also affects the more tonically active vasoconstrictor system in glabrous and nonglabrous skin during cooling, healthy males (n = 9) underwent afternoon sessions of whole body skin temperature (Tsk) cooling (water‐perfused suits) after oral melatonin (Mel; 3 mg) or placebo (Cont). Cutaneous vascular conductance (CVC) was calculated from SkBF (laser Doppler flowmetry) and non‐invasive blood pressure. Baseline Tint was lower in Mel than in Cont (P < 0.01). During progressive reduction of Tsk from 35°C to 32°C, forearm CVC was first significantly reduced at Tsk of 34.33 ± 0.01°C (P < 0.05) in Cont. In contrast, CVC in Mel was not significantly reduced until Tsk reached 33.33 ± 0.01°C (P < 0.01). The decrease in forearm CVC in Mel was significantly less than in Cont at Tsk of 32.66 ± 0.01°C and lower (P < 0.05). In Mel, palmar CVC was significantly higher than in Cont above Tsk of 33.33 ± 0.01°C, but not below. Thus exogenous melatonin blunts reflex vasoconstriction in nonglabrous skin and shifts vasoconstrictor system control to lower Tint. It provokes vasodilation in glabrous skin but does not suppress the sensitivity to falling Tsk. These findings suggest that by affecting the vasoconstrictor system, melatonin has a causal role in the nocturnal changes in body temperature and its control.

Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca2+ and Voltage Activated (BK) Potassium Channel

Ion channels are integral membrane proteins that regulate membrane potentials and signaling of cells in response to various stimuli. The patch-clamp technique enables the study of single channels or a population of channels. The macroscopic recording approaches are powerful in revealing population-averaged behaviors of channels both under basal conditions and in response to various stimuli, modulators and drugs. On their own, however, these approaches can be insufficient for determinations of channel gating mechanisms as they do not accurately report channel open probabilities below 10(-2) to 10(-3). This obstacle can be overcome with the use of single-channel recording techniques. Single-channel recording techniques can be applied to one or a few channels to estimate P o over a larger range than macroscopic recordings. The combination of heterologous overexpression of ion channels with macroscopic and single-channel recordings can be applied to hundreds of channels to estimate P o between 1 and 10(-8). Here, we describe practical approaches of single-channel recordings that our laboratory utilizes. We also provide examples where the combined macroscopic and single channel approach can be employed to study gating mechanisms of the BK type, large conductance, Ca(2+) and voltage activated potassium channel in a mammalian expression system. The techniques presented should be generally applicable to the studies of ion channels in heterologous expression systems.