Lower hypoxic ventilatory response in smokers compared to non-smokers during abstinence from cigarettes.

BACKGROUND: Carotid body O2-chemosensitivity determines the hypoxic ventilatory response (HVR) as part of crucial regulatory reflex within oxygen homeostasis. Nicotine has been suggested to attenuate HVR in neonates of smoking mothers. However, whether smoking affects HVR in adulthood has remained unclear and probably blurred by acute ventilatory stimulation through cigarette smoke. We hypothesized that HVR is substantially reduced in smokers when studied after an overnight abstinence from cigarettes i.e. after nicotine elimination. METHODS: We therefore determined the isocapnic HVR of 23 healthy male smokers (age 33.9 ± 2.0 years, BMI 24.2 ± 0.5 kg m-2, mean ± SEM) with a smoking history of >8 years after 12 h of abstinence and compared it to that of 23 healthy male non-smokers matched for age and BMI. RESULTS: Smokers and non-smokers were comparable with regard to factors known to affect isocapnic HVR such as plasma levels of glucose and thiols as well as intracellular levels of glutathione in blood mononuclear cells. As a new finding, abstinent smokers had a significantly lower isocapnic HVR (0.024 ± 0.002 vs. 0.037 ± 0.003 l min-1 %-1BMI-1, P = 0.002) compared to non-smokers. However, upon re-exposure to cigarettes the smokers' HVR increased immediately to the non-smokers' level. CONCLUSIONS: This is the first report of a substantial HVR reduction in abstinent adult smokers which appears to be masked by daily smoking routine and may therefore have been previously overlooked. A low HVR may be suggested as a novel link between smoking and aggravated hypoxemia during sleep especially in relevant clinical conditions such as COPD.

Heart failure and carotid body chemoreception.

There is substantial evidence to implicate a role of the carotid body (CB) chemoreflex in sympathetic and breathing dysregulation in several cardio-respiratory diseases, drawing renewed interest in its potential implications for clinical treatment and management. Evidence from both chronic heat failure (CHF) patients and animal models indicates that the CB chemoreflex is enhanced in CHF and contributes to the tonic elevation in sympathetic nerve activity (SNA) and periodic breathing associated with the disease. Although this maladaptive change likely derives from altered function at all levels of the reflex arc, a change in afferent function of the CB is likely to be a main driving force. This review will focus on recent advances in our understanding of the physiological mechanisms that alter CB function in CHF and their potential translational impact on treatment of CHF.

Autonomic innervation of the carotid body as a determinant of its sensitivity: implications for cardiovascular physiology and pathology.

The motivation for this review comes from the emerging complexity of the autonomic innervation of the carotid body (CB) and its putative role in regulating chemoreceptor sensitivity. With the carotid bodies as a potential therapeutic target for numerous cardiorespiratory and metabolic diseases, an understanding of the neural control of its circulation is most relevant. Since nerve fibres track blood vessels and receive autonomic innervation, we initiate our review by describing the origins of arterial feed to the CB and its unique vascular architecture and blood flow. Arterial feed(s) vary amongst species and, unequivocally, the arterial blood supply is relatively high to this organ. The vasculature appears to form separate circuits inside the CB with one having arterial venous anastomoses. Both sympathetic and parasympathetic nerves are present with postganglionic neurons located within the CB or close to it in the form of paraganglia. Their role in arterial vascular resistance control is described as is how CB blood flow relates to carotid sinus afferent activity. We discuss non-vascular targets of autonomic nerves, their possible role in controlling glomus cell activity, and how certain transmitters may relate to function. We propose that the autonomic nerves sub-serving the CB provide a rapid mechanism to tune the gain of peripheral chemoreflex sensitivity based on alterations in blood flow and oxygen delivery, and might provide future therapeutic targets. However, there remain a number of unknowns regarding these mechanisms that require further research that is discussed.

Peripheral chemoreceptors determine the respiratory sensitivity of central chemoreceptors to CO(2).

We assessed the contribution of carotid body chemoreceptors to the ventilatory response to specific CNS hypercapnia in eight unanaesthetized, awake dogs. We denervated one carotid body (CB) and used extracorporeal blood perfusion of the reversibly isolated remaining CB to maintain normal CB blood gases (normoxic, normocapnic perfusate), to inhibit (hyperoxic, hypocapnic perfusate) or to stimulate (hypoxic, normocapnic perfusate) the CB chemoreflex, while the systemic circulation, and therefore the CNS and central chemoreceptors, were exposed consecutively to four progressive levels of systemic arterial hypercapnia via increased fractional inspired CO(2) for 7 min at each level. Neither unilateral CB denervation nor CB perfusion, per se, affected breathing. Relative to CB control conditions (normoxic, normocapnic perfusion), we found that CB chemoreflex inhibition decreased the slope of the ventilatory response to CNS hypercapnia in all dogs to an average of 19% of control values (range 0-38%; n = 6), whereas CB chemoreflex stimulation increased the slope of the ventilatory response to CNS hypercapnia in all dogs to an average of 223% of control values (range 204-235%; n = 4). We conclude that the gain of the CNS CO(2)/H(+) chemoreceptors in dogs is critically dependent on CB afferent activity and that CNS-CB interaction results in hyperadditive ventilatory responses to central hypercapnia.

Ultrastructure of the carotid body.

An electron microscope investigation was made of the carotid body in the cat and the rabbit. In thin-walled blood vessels the endothelium was fenestrated. Larger vessels were surrounded by a layer of smooth muscle fibers. Among the numerous blood vessels lay groups of cells of two types covered by basement membranes. Aggregates of Type I cells were invested by Type II cells, though occasionally cytoplasmic extensions were covered by basement membrane only. Type I cells contained many electron-opaque cored vesicles (350 to 1900 A in diameter) resembling those in endocrine secretory cells. Type II cells covered nerve endings terminating on Type I cells and enclosed nerve fibers in much the same manner as Schwann cells. The nerve endings contained numerous microvesicles ( approximately 500 A in diameter), mitochondria, glycogen granules, and a few electron-opaque cored vesicles. Junctions between nerve endings and Type I cells were associated with regions of increased density in both intercellular spaces and the adjoining cytoplasm. Cilia of the 9 + 0 fibril pattern were observed in Type I and Type II cells and pericytes. Nonmyelinated nerve fibers, often containing microvesicles, mitochondria, and a few electron-opaque cored vesicles (650 to 1000 A in diameter) were present in Schwann cells, many of which were situated close to blood vessels Ganglion cells near the periphery of the gland, fibrocytes, and segments of unidentified cells were also seen. It was concluded that, according to present concepts of the structure of nerve endings, those endings related to Type I cells could be efferent or afferent.

Expression and immunolocalization of endothelin peptides and its receptors, ETA and ETB, in the carotid body exposed to chronic intermittent hypoxia.

Increased levels of endothelin-1 (ET-1) in the carotid body (CB) contribute to the enhancement of chemosensory responses to acute hypoxia in cats exposed to chronic intermittent hypoxia (CIH). However, it is not known if the ET receptor types A (ETA-R) and B (ETB-R) are upregulated. Thus, we studied the expression and localization of ETA-R and ETB-R using Western blot and immunohistochemistry (IHC) in CBs from cats exposed to cyclic hypoxic episodes, repeated during 8 hr for 4 days. In addition, we determined if ET-1 is expressed in the chemoreceptor cells using double immunofluorescence for ET-1 and tyrosine hydroxylase (TH). We found that ET-1 expression was ubiquitous in the blood vessels and CB parenchyma, although double ET-1 and TH-positive chemoreceptor cells were mostly found in the parenchyma. ETAR was expressed in most chemoreceptor cells and blood vessels of the CB vascular pole. ETB-R was expressed in chemoreceptor cells, parenchymal capillaries, and blood vessels of the vascular pole. CIH upregulated ETB-R expression by approximately 2.1 (Western blot) and 1.6-fold (IHC) but did not change ETA-R expression. Present results suggest that ET-1,ETA-R, and ETB-R are involved in the enhanced CB chemosensory responses to acute hypoxia induced by CIH.

Paradoxical effect of phentolamine on aqueous flow in the rabbit.

PURPOSE: The aim of this study was to assess the effects of acute systemic, nonselective alpha-adrenergic blockade on aqueous flow. METHODS: This study used pentobarbital-anesthetized rabbits (n=7), in which the following parameters were measured: mean arterial pressure, carotid blood flow, heart rate, intraocular pressure (IOP), orbital venous pressure (OVP), ciliary blood flow, and aqueous flow (AqFlow). Measurements were made before and after an intravenous administration of phentolamine (0.1 mg/kg). RESULTS: Phentolamine caused significant decreases in IOP -23%+/-2%; P<0.01), OVP (-28%+/-12%; P<0.05), and AqFlow (-33%+/-6%; P<0.01). The other parameters were not significantly altered. The rapidity of the OVP and IOP responses were noteworthy, being essentially complete 60 s after the phentolamine injection. CONCLUSIONS: A subpressor dose of phentolamine has complex effects on ocular hydrodynamics. The initial IOP decrease is too fast to be explained by aqueous dynamics or ocular rigidity, and so is most likely a result of the disgorgement of choroidal blood volume caused by decreased venous pressure outside the eye. The more prolonged ocular hypotensive effect is explained by the decrease in AqFlow, and perhaps a decrease in episcleral venous pressure or increase in uveoscleral outflow. However, the inhibition of aqueous production is odd, as lost prejunctional inhibition of norepinephrine release and unopposed beta-receptor activation should have increased aqueous production.

[Effects of tissue inhibitor of matrix metalloproteinases-4 on the activities of matrix metalloproteinases and collagen of artery: experiment with rats].

OBJECTIVE: To investigate the effects of tissue inhibitor of matrix metalloproteinases-4 (TIMP-4) on the activities of matrix metalloproteinases (MMPs) and the collagen deposition. METHODS: Vascular smooth muscle cells (VSMCs) of rat thoracic aorta were cultured and divided into 3 groups: 2 groups to be transfected with adenovirus vector containing green fluorescence protein (AdGFP) or adenovirus vector containing TIMP-4 (AdTIMP-4), and one group un-transfected. Zymography and reverse zymography were used to detect the activities of MMP-2, MMP-9 and TIMP-4 in the supernatants. Male Wistar rats underwent balloon injury of common carotid artery and then divided into 3 groups: pure injury group, AdGFP transfection group, and AdTIMP-4. transfection group. Four and 28 days later 3 and 6 rats were killed in each group respectively to undergo microscopy and examination of the activities of MMP-2, MMP-9, and TIMP-4, and collagen quantity. RESULTS: The MMP-2 activity in the VSMC culture fluid supernatant of the AdTIMP-4 group was decreased dose-dependently, however, the activity of MMP-9 did not changed significantly among the 3 groups. Bands of activated MMP-2 and MMP-9 could not be examined in the normal vessel tissues. Four hours after the injury, the activity of MMP-2 was significantly increased in the pure injury group and AdGFP group, however, was significantly decreased in the AdTIMP-4 group. Four days later no MMP activity could be detected in either group. The neoformation of tunica intima was inhibited by 66% in the AdTIMP-4 group, The collagen quantity per vessel cell was 12.1 +/- 1.0 in the AdGFP group, not significantly different from that of in the AdTIMP-4 group (11.9 +/- 1, P > 0.05), and the collagen quantity per tunica adventitia cell in the AdTIMP-4 group was 118 +/- 13, not significantly different from that of the pure injury group (135 +/- 11, P > 0.05). CONCLUSION: The regulation of MMP/TIMP balance by TIMP-4 may control the metabolism of collagen and play an important role in the vascular repair process.

Hypertension: a problem of organ blood flow supply-demand mismatch.

This review introduces a new hypothesis that sympathetically mediated hypertensive diseases are caused, in the most part, by the activation of visceral afferent systems that are connected to neural circuits generating sympathetic activity. We consider how organ hypoperfusion and blood flow supply-demand mismatch might lead to both sensory hyper-reflexia and aberrant afferent tonicity. We discuss how this may drive sympatho-excitatory-positive feedback and extend across multiple organs initiating, or at least amplifying, sympathetic hyperactivity. The latter, in turn, compounds the challenge to sufficient organ blood flow through heightened vasoconstriction that both maintains and exacerbates hypertension.

Immediate and long-term responses of the carotid body to high altitude.

High altitude and the decreased environmental oxygen pressure have both immediate and chronic effects on the carotid body. An immediate effect is to limit the oxygen available for mitochondrial oxidative phosphorylation, and this leads to increased activity on the afferent nerves leading to the brain. In the isolated carotid body preparation, the afferent nerve activity depends on the ratio of carbon monoxide (CO), an inhibitor of respiratory chain function, to oxygen. The CO-induced increase in afferent neural activity is reversed by light, and the wavelength dependence of this reversal shows that the site of CO (and therefore oxygen) interaction is cytochrome a3 of the mitochondrial respiratory chain. Thus, primary sensing of ambient oxygen pressure is through the oxygen dependence of mitochondrial oxidative phosphorylation. The conductance of ion channels in the cellular membranes may also be sensitive to oxygen pressure and, through this, modulate the sensitivity to oxygen pressure. Longer-term exposure to high altitude results in progressive changes in the carotid body that involve several mechanisms, including cellular energy metabolism and hypoxia inducible factor-1alpha (HIF-1alpha). These changes begin within minutes of exposure, but progress such that chronic exposure results in morphological and biochemical alterations in the carotid body, including enlarged cells, increased catecholamine levels, altered cellular appearance, and others. In the chronically adapted carotid body, responses to acute changes in oxygen pressure are enhanced. The adaptive changes due to chronic hypoxia are largely reversed upon return to lower altitudes.

Cellular and molecular mechanisms associated with carotid body adaptations to chronic hypoxia.

Chronic hypoxia leads to adaptations in the respiratory system manifested as a persistent increase in resting ventilation, termed ventilatory acclimatization to hypoxia (VAH). Increased afferent nerve activity from carotid bodies and the ensuing reflex activation of ventilation are critical for eliciting VAH. In this review we highlight recent information on the cellular and molecular mechanisms associated with chronic hypoxia-induced functional and structural changes in the carotid body. Chronic hypoxia leads to hypersensitivity of the carotid bodies and induces morphological changes, including enlargement of the organ, hyperplasia of glomus cells, and neovascularization. Enhanced hypoxic sensitivity is due to alterations in ion current densities, as well as changes in neurotransmitter dynamics and recruitment of additional neuromodulators (endothelin- 1, ET-1) in glomus cells. Morphological alterations are in part due to upregulation of growth factors (e.g., VEGF). VAH is markedly attenuated in mice partially deficient in HIF-1 transcription factor, which regulates several downstream genes, including VEGF, ET-1, and Ca(2+) channels. The finding that VAH is also blunted in mice deficient in the fosB gene led to the suggestion that the magnitude and time course of VAH depend on complex interactions between more than one transcription factor, resulting in coordinated regulation of several downstream genes and their protein products.

Mitochondrial function and carotid body transduction.

Carotid body chemoreceptors respond to a decrease in arterial oxygen tension by increasing spiking activity on the sinus nerve. Our understanding of the oxygen-transducing ability of the organ arose from studies in the 1930s intended to understand how metabolic poisons stimulated breathing. Since that time, an intimate link between energy state and hypoxia sensing has been assumed and forms the basis of the metabolic hypothesis of oxygen sensing. This hypothesis is supported by studies demonstrating a loss of mitochondrial potential in carotid body cells at oxygen tensions that cause no change in cells from other tissues. Although the nature of the coupling between mitochondrial function and nerve excitation remains unresolved, experimental evidence supports roles for (1) release of mitochondrial calcium stores, (2) modulation of membrane channels that are linked to mitochondrial complexes I and IV, and (3) generation of signaling intermediates, such as reactive oxygen species (ROS) from complex I and III of the electron transport chain. If the mitochondrion is the oxygen-sensing site for peripheral chemoreceptors, then there exists the potential ability to manipulate, perhaps pharmacologically, the sensing function by alterations in expression of uncoupler proteins or chemicals that can alter the affinity of cytochrome oxidase for oxygen. Such manipulation may be useful for the treatment of hypoventilation syndromes or high altitude accommodation.

Endothelial vesiculo-vacuolar organelles, pockets and multi-layered fenestrated lamellae in the capillaries of the mouse carotid body.

Fenestrated capillaries represent the basic structural unit in the carotid body. They mediate a characteristic hyperpermeability state in this organ. Endothelial fenestrae and plasmalemmal vesicles are of particular importance in this respect. The present electron microscopic study of the capillaries of the mouse carotid body demonstrates prominent endothelial cell structures that are suggested to be closely related to endothelial fenestrae and plasmalemmal vesicles. These structures include: (1) Vesiculo-vacuolar organelles formed by fusion and intercommunication of vesicles and vacuoles of variable dimensions. (2) Pockets in the form of fenestrated membrane-bound vacuoles that communicate either with the capillary lumen, pericapillary space or both via multiple apertures or fenestrae. (3) Multi-layered fenestrated Lamellae where the endothelial cytoplasm is divided into multiple attenuated sheets provided with several fenestrae. The latter two structures are preferentially located in the thick perinuclear region of the endothelial cell. Their fenestrae are always distributed in linear series and show close similarity to the usual chains of fenestrae in the attenuated periphery of the endothelial cells. The individual apertures of the fenestrated vacuoles and multi-layered fenestrated lamellae are closely similar to the stomata of fully opened plasmalemmal vesicles suggesting a relationship between them. Morphological and morphometrical analysis of a series of fenestrae belonging to these structures revealed that they are identical to the usual chains of fenestrae in the attenuated periphery of the endothelial cells.

The vasculature of the carotid body.

A histological and electron microscopical study was carried out on the vasculature of the carotid bodies in seven subjects coming to necropsy. None of these had suffered from chronic hypoxaemia or systemic hypertension during life and none had hypertrophy of the right or left ventricle. The vasculature of the carotid bodies showed three distinct components. There was a proximal portion, comprising elastic interlobular arteries, which had a wall in which elastic tissue predominated and in which many nerve fibrils could be demonstrated. This is considered to be baroreceptor in nature. There was an intermediate portion comprising muscular intralobular arterioles which are believed to be capable of controlling the level of blood supply to the parenchyma of the carotid bodies. Finally there are glomic capillaries surrounded by elongated pericytes and sustentacular cells. One or other of these elongated cells is thought to be responsible for the carotid body hyperplasia which is associated with systemic hypertension and states of chronic hypoxaemia.

Glomic cells and blood vessels in the hyperplastic carotid bodies of spontaneously hypertensive rats.

Carotid bodies from 21 normotensive Wistar albino rats were compared with those from 20 spontaneously hypertensive rats of the Okamoto strain. From serial histological sections the volume of the carotid bodies was estimated by Simpson's rule. Differential and absolute cell counts were also performed on the chief and elongated cells which surround them. The glomic vasculature was examined by light and electron microscopy. Although the carotid bodies of Okamoto rats were nearly three times as large as those of Wistar rats of comparable body weight, there was no difference in the proportion of the two types of cell. The organisation of glomic cells was also similar in the two strains. The main carotid body artery consists of a muscular tube with a valve-like cushion at its orifice. In the Okamoto rats branches of this artery were occluded by intimal proliferations of myofibroblasts embedded within a copious, loose matrix of acid mucopolysaccharide ground substance. These proliferations appeared to originate from intimal pads situated at the origins of many glomic arteries and arterioles. These findings are in sharp contrast to those in the hyperplastic human carotid body.

Nerve branching and terminal arborizations in the carotid body of the cat. A light microscopic study following anterograde injury filling of carotid nerve axons with horseradish peroxidase.

The terminal arborizations of carotid nerve axons within the carotid body of the cat were densely filled with horseradish peroxidase and studied under the light microscope. Two types of terminal arborizations were found in contact with glomus (type I) cells. The axons differed principally in the wealth of terminal swellings. The largest and most numerous type of arborization consisted of one to several clusters of terminals of variable size and shape arising from a single fiber and distributed in a rather ellipsoidal domain of about 9,000 microns 3 for each cluster. Thus, these arborizations might be in close relation with 20-60 glomus cells. The second type of arborization had substantially fewer terminal swellings, occupying a smaller volume and probably contacted significantly less glomus cells. Both kinds of axons had small rounded and large calyciform endings. The larger arborizations were derived consistently from larger fibers than those which produced the smaller arborizations. The results suggest that the carotid nerve axons generate two types of arborizations within the carotid body. Thus, glomus cells potentially can contact two classes of afferent fibers. The functional significance of a dual chemoreceptor innervation of the carotid body is discussed.

Morphological changes in the rat carotid body 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia.

Morphological changes in the rat carotid bodies 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia (10% O2 for 8 weeks) were examined by means of morphometry and immunohistochemistry. The rat carotid bodies after 8 weeks of hypoxic exposure were enlarged several fold with vascular expansion. The carotid bodies 1 and 2 weeks after the termination of 8 weeks of hypoxic exposure were diminished in size, although their diameter remained larger than the normoxic controls. The expanded vasculature in chronically hypoxic carotid bodies returned to the normoxic control state. In the carotid bodies 1 week after the termination of chronic hypoxia, the density of NPY fibers was remarkably increased and that of VIP fibers was dramatically decreased in comparison with the density in chronically hypoxic carotid bodies. In the carotid bodies 2 and 4 weeks after the termination of hypoxia, the density of SP and CGRP fibers was gradually increased. In the carotid bodies 8 weeks after the termination of hypoxia, the appearance of the carotid body returned to a nearly normoxic state, and the density of SP, CGRP, VIP, and NPY fibers also recovered to that of normoxic controls. These results suggest that the morphological changes in the recovering carotid bodies start at a relatively early period after the termination of chronic hypoxia, and a part of these processes may be under the control of peptidergic innervation.

Chronic hypoxia-induced morphological and neurochemical changes in the carotid body.

The carotid body (CB) plays an important role in the control of ventilation. Type I cells in CB are considered to be the chemoreceptive element which detects the levels of PO(2), PCO(2), and [H(+)] in the arterial blood. These cells originate from the neural crest and appear to retain some neuronal properties. They are excitable and produce a number of neurochemicals. Some of these neurochemicals, such as dopamine and norepinephrine, are considered to be primarily inhibitory to CB function and others, such as adenosine triphosphate, acetylcholine, and endothelin, are thought to be primarily excitatory. Chronic hypoxia (CH) induces profound morphological as well as neurochemical changes in the CB. CH enlarges the size of CB and causes hypertrophy and mitosis of type I cells. Also, CH changes the vascular structure of CB, including inducing marked vasodilation and the growth of new blood vessels. Moreover, CH upregulates certain neurochemical systems within the CB, e.g., tyrosine hydroxylase and dopaminergic activity in type I cells. There is also evidence that CH induces neurochemical changes within the innervation of the CB, e.g., nitric oxide synthase. During CH the sensitivity of the CB chemoreceptors to hypoxia is increased but the mechanisms by which the many CH-induced structural and neurochemical changes affect the sensitivity of CB to hypoxia remains to be established.

Measurement of carotid body blood flow in cats by use of radioactive microspheres.

To resolve the controversy regarding carotid body blood flow, we used the radioactive microsphere technique for determination of tissue blood flow. We also measured the blood flow to several other tissues in the cat. Blood flow experiments were performed on 13 cats that were anesthetized, paralyzed, and mechanically ventilated with air. Different numbers of differently labeled 9-, 15-, and 25-micron microspheres were injected via a catheter into the left atrium. It was determined that one injection of 5 x 10(6) 15-micron microspheres was appropriate for the determination of carotid body blood flow. Flows to the carotid bodies and other organs by use of this protocol were as follows (ml.min-1.100 g-1, means +/- SE): carotid bodies, 1,417 +/- 143; adrenal glands, 406 +/- 89; left kidney, 355 +/- 69; right kidney, 375 +/- 74; heart, 201 +/- 39; liver 81 +/- 14; pancreas, 80 +/- 21; superior cervical ganglia, 62 +/- 9; carotid artery wall, 2.4 +/- 1.1. The blood flow to the carotid bodies was the highest for any organ. This measurement provides new evidence that tissue blood flow to the carotid body is very high. This high flow is consistent with the prompt physiological reflex functions of the carotid body.