Dean L. Kellogg

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.

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (μD) is the only tool available that explicitly provides data on the extracellular space. Although μD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of μD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of μD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on μD as a tool in drug research and development.

Local thermal control of the human cutaneous circulation.

The level of skin blood flow is subject to both reflex thermoregulatory control and influences from the direct effects of warming and cooling the skin. The effects of local changes in temperature are capable of maximally vasoconstricting or vasodilating the skin. They are brought about by a combination of mechanisms involving endothelial, adrenergic, and sensory systems. Local warming initiates a transient vasodilation through an axon reflex, succeeded by a plateau phase due largely to nitric oxide. Both phases are supported by sympathetic transmitters. The plateau phase is followed by the die-away phenomenon, a slow reversal of the vasodilation that is dependent on intact sympathetic vasoconstrictor nerves. The vasoconstriction with local skin cooling is brought about, in part, by a postsynaptic upregulation of α(2c)-adrenoceptors and, in part, by inhibition of the nitric oxide system at at least two points. There is also an early vasodilator response to local cooling, dependent on the rate of cooling. The mechanism for that transient vasodilation is not known, but it is inhibited by intact sympathetic vasoconstrictor nerve function and by intact sensory nerve function.

Cutaneous vasodilator and vasoconstrictor mechanisms in temperature regulation.

In this review, we focus on significant developments in our understanding of the mechanisms that control the cutaneous vasculature in humans, with emphasis on the literature of the last half-century. To provide a background for subsequent sections, we review methods of measurement and techniques of importance in elucidating control mechanisms for studying skin blood flow. In addition, the anatomy of the skin relevant to its thermoregulatory function is outlined. The mechanisms by which sympathetic nerves mediate cutaneous active vasodilation during whole body heating and cutaneous vasoconstriction during whole body cooling are reviewed, including discussions of mechanisms involving cotransmission, NO, and other effectors. Current concepts for the mechanisms that effect local cutaneous vascular responses to local skin warming and cooling are examined, including the roles of temperature sensitive afferent neurons as well as NO and other mediators. Factors that can modulate control mechanisms of the cutaneous vasculature, such as gender, aging, and clinical conditions, are discussed, as are nonthermoregulatory reflex modifiers of thermoregulatory cutaneous vascular responses.

Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans

Nitric oxide (NO) participates in the cutaneous vasodilation caused by increased local skin temperature (Tloc) and whole body heat stress in humans. In forearm skin, endothelial NO synthase (eNOS) participates in vasodilation due to elevated Tloc and neuronal NO synthase (nNOS) participates in vasodilation due to heat stress. To explore the relative roles and interactions of these isoforms, we examined the effects of a relatively specific eNOS inhibitor, N(omega)-amino-l-arginine (LNAA), and a specific nNOS inhibitor, N(omega)-propyl-l-arginine (NPLA), both separately and in combination, on skin blood flow (SkBF) responses to increased Tloc and heat stress in two protocols. In each protocol, SkBF was monitored by laser-Doppler flowmetry (LDF) and mean arterial pressure (MAP) by Finapres. Cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). Intradermal microdialysis was used to treat one site with 5 mM LNAA, another with 5 mM NPLA, a third with combined 5 mM LNAA and 5 mM NPLA (Mix), and a fourth site with Ringer only. In protocol 1, Tloc was controlled with combined LDF/local heating units. Tloc was increased from 34 degrees C to 41.5 degrees C to cause local vasodilation. In protocol 2, after a period of normothermia, whole body heat stress was induced (water-perfused suits). At the end of each protocol, all sites were perfused with 58 mM nitroprusside to effect maximal vasodilation for data normalization. In protocol 1, at Tloc = 34 degrees C, CVC did not differ between sites (P > 0.05). LNAA and Mix attenuated CVC increases at Tloc = 41.5 degrees C to similar extents (P < 0.05, LNAA or Mix vs. untreated or NPLA). In protocol 2, in normothermia, CVC did not differ between sites (P > 0.05). During heat stress, NPLA and Mix attenuated CVC increases to similar extents, but no significant attenuation occurred with LNAA (P < 0.05, NPLA or Mix vs. untreated or LNAA). In forearm skin, eNOS mediates the vasodilator response to increased Tloc and nNOS mediates the vasodilator response to heat stress. The two isoforms do not appear to interact during either response.

Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo

Nitric oxide (NO) participates in locally mediated vasodilation induced by increased local skin temperature (T(loc)) and in sympathetically mediated vasodilation during whole body heat stress. We hypothesized that endothelial NOS (eNOS) participates in the former, but not the latter, response. We tested this hypothesis by examining the effects of the eNOS antagonist N(G)-amino-l-arginine (l-NAA) on skin blood flow (SkBF) responses to increased T(loc) and whole body heat stress. Microdialysis probes were inserted into forearm skin for drug delivery. One microdialysis site was perfused with l-NAA in Ringer solution and a second site with Ringer solution alone. SkBF [laser-Doppler flowmetry (LDF)] and blood pressure [mean arterial pressure (MAP)] were monitored, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF / MAP). In protocol 1, T(loc) was controlled with LDF/local heating units. T(loc) initially was held at 34 degrees C and then increased to 41.5 degrees C. In protocol 2, after a normothermic period, whole body heat stress was induced (water-perfused suits). At the end of both protocols, 58 mM sodium nitroprusside was perfused at both microdialysis sites to cause maximal vasodilation for data normalization. In protocol 1, CVC at 34 degrees C T(loc) did not differ between l-NAA-treated and untreated sites (P > 0.05). Local skin warming to 41.5 degrees C T(loc) increased CVC at both sites. This response was attenuated at l-NAA-treated sites (P < 0.05). In protocol 2, during normothermia, CVC did not differ between l-NAA-treated and untreated sites (P > 0.05). During heat stress, CVC rose to similar levels at l-NAA-treated and untreated sites (P > 0.05). We conclude that eNOS is predominantly responsible for NO generation in skin during responses to increased T(loc), but not during reflex responses to whole body heat stress.

Neuronal nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo.

The physiological roles of constituitively expressed nitric oxide synthase (NOS) isoforms in humans, in vivo, are unknown. Cutaneous vasodilatation during both central nervous system‐mediated, thermoregulatory reflex responses to whole‐body heat stress and during peripheral axon reflex‐mediated, local responses to skin warming in humans depend on nitric oxide (NO) generation by constituitively expressed NOS of uncertain isoform. We hypothesized that neuronal NOS (nNOS, NOS I) effects cutaneous vasodilatation during whole‐body heat stress, but not during local skin warming. We examined the effects of the nNOS inhibitor 7‐nitroindazole (7‐NI) administered by intradermal microdialysis on vasodilatation induced by whole‐body heat stress or local skin warming. Skin blood flow (SkBF) was monitored by laser–Doppler flowmetry (LDF). Blood pressure (MAP) was monitored and cutaneous vascular conductance calculated (CVC = LDF/MAP). In protocol 1, whole‐body heat stress was induced with water‐perfused suits. In protocol 2, local skin warming was induced through local warming units at LDF sites. At the end of each protocol, 56 mm sodium nitroprusside was perfused at microdialysis sites to raise SkBF to maximal levels for data normalization. 7‐NI significantly attenuated CVC increases during whole‐body heat stress (P < 0.05), but had no effect on CVC increases induced by local skin warming (P > 0.05). These diametrically opposite effects of 7‐NI on two NO‐dependent processes verify selective nNOS antagonism, thus proving that the nNOS isoform affects NO increases and hence vasodilatation during centrally mediated, reflex responses to whole‐body heat stress, but not during locally mediated, axon reflex responses to local skin warming. We conclude that the constituitively expressed nNOS isoform has distinct physiological roles in cardiovascular control mechanisms in humans, in vivo.

Evidence for a Role for Vasoactive Intestinal Peptide in Active Vasodilatation in the Cutaneous Vasculature of Humans

Active vasodilatation (AVD) in human, non‐glabrous skin depends on functional cholinergic fibres but not on acetylcholine (ACh). We tested whether AVD is a redundant system in which ACh and vasoactive intestinal polypeptide (VIP) are co‐released from cholinergic nerves. (1) We administered VIP by intradermal microdialysis to four discrete areas of skin in the presence of different levels of the VIP receptor antagonist, VIP(10−28), also delivered by microdialysis. Skin blood flow (SkBF) was continuously monitored by laser Doppler flowmetry (LDF). Mean arterial pressure (MAP) was measured non‐invasively and cutaneous vascular conductance (CVC) calculated as LDF/MAP. Subjects were supine and wore water‐perfused suits to control whole‐body skin temperature (Tsk) at 34 °C. Concentrations of 54 μm, 107 μM, or 214 μM VIP(10−28) were perfused via intradermal microdialysis at 2 μl min−1 for approximately 1 h. Then 7.5 μM VIP was added to the perfusate containing VIP(10−28) at the three concentrations or Ringer solution and perfusion was continued for 45‐60 min. At the control site, this level of VIP caused approximately the vasodilatation typical of heat stress. All VIP(10−28)‐treated sites displayed an attenuated dilatation in response to the VIP. The greatest attenuation was observed at the site that received 214 μM VIP(10−28) (P < 0.01). (2) We used 214 μM VIP(10−28) alone and with the iontophoretically administered muscarinic receptor antagonist atropine (400 μA cm−2, 45 s, 10 mM) in heated subjects to test the roles of VIP and ACh in AVD. Ringer solution and 214 μM VIP(10−28) were each perfused at two sites, one of which in each case was pretreated with atropine. After 1 h of VIP(10−28) treatment, individuals underwent 45−60 min of whole‐body heating (Tsk to 38.5 °C). VIP(10−28), alone or in combination with atropine, attenuated the increase in CVC during heat stress, suggesting an important role for VIP in AVD.

Intradermal microdialysis: kinetics of iontophoretically delivered propranolol in forearm dermis.

Intradermal microdialysis permits us to measure the concentration in dermis of drugs applied to the skin. Microdialysis is especially efficient in sampling water-soluble molecules. Consequently, it appears particularly suitable to study current based delivery systems like iontophoresis that deliver ions or highly polar molecules. The purpose of this work was to evaluate the adequacy of a skin microdialysis technique to characterize and quantify the dermatopharmacokinetics of iontophoretically delivered propranolol in the dermis of healthy human volunteers. Linear microdialysis probes were inserted in the subjects forearm skin and an iontophoresis device was installed above them. Constant current was applied for two periods of 1 h each separated by a 1-h interval. Dialysate samples were collected every 6 min for 4.4 h and analyzed by HPLC. Probes were always placed in the dermis as measured by ultrasonography. Propranolol was detectable in the dialysate. It was possible to build detailed concentration vs. midtime profiles that mirrored the current applied. Elimination rate from the dermis had first-order kinetics and was similar in all subjects. Quantification of the absorption process, indexed by lag-time and area under the concentration curve showed a high inter- and intrasubject variability that did not correlate with probe depth.

Effects of alternating current iontophoresis on drug delivery

OBJECTIVE The duration of direct current (DC) iontophoresis is limited to 10- to 15-minute periods because of electrochemical burns from hydrogen and hydroxide ions generated by the DC current. A new iontophoretic device, the Lectro Patch, uses a low-frequency alternating current (AC). AC current is theorized to generate H+ ions during one phase and OH- when the current reverses polarity, thus possibly neutralizing pH changes and avoiding burns. This study examined this possibility and evaluated drug delivery with AC iontophoresis, using hydroxocobalamin. DESIGN A known amount of hydroxocobalamin dissolved in 6mL of water was loaded in Lectro Patches, two of which were then taped on the forearms of 10 patient volunteers. One patch was activated to deliver drug by AC iontophoresis. The second patch was not activated and served as a control for delivery by diffusion. Trials were run for 2 and 4 hours, with both 1,000 micrograms/mL and 2,000 micrograms/mL concentrations. SETTING Study was conducted with inpatients in an extended care setting using volunteers. MAIN OUTCOME MEASURES Amounts of hydroxocobalamin remaining in the Lectro Patches after iontophoresis were assayed by spectrophotometry. Data were analyzed by ANOVA. RESULTS No burns occurred. Significantly greater losses occurred with 4 hours of iontophoresis than with 2 hours (p < 0.05). There was no significant effect of changing the concentration of hydroxocobalamin. CONCLUSIONS AC iontophoresis avoids electrochemical burns; charged drugs can be delivered by AC iontophoresis; and delivery of drug increases with duration of application.