The occurrence and distribution of certain polypeptides within the human carotid body

Summary Both carotid bodies from 26 patients coming to necropsy were fixed in 10% neutral buffered formalin and sections 4 m thick were stained for various peptides by use of the immunogold technique. The results show that the human carotid body contains met- and leu-enkephalin, substance P, vasoactive intestinal peptide (VIP), neurotensin and bombesin. The distribution of these six peptides within the carotid body differs. Thus met- and leu-enkephalin are both present predominantly within glomic chief cells but with a marked tendency to favour the dark variant of these cells. Substance P and VIP both show a weak immunoreactivity in comparison to the enkephalins and are present in all three variants of chief cell. Neurotensin shows the weakest immunoreactivity of all and is restricted to a few glomic chief cells in a minority of cases. Bombesin also shows a weak immunoreactivity in glomic chief cells but a strong reaction in glomic arteries and arterioles. In these vessels bombesin appears to be confined to smooth muscle cells in the media but we cannot say whether it is secreted by them or merely bound to receptor sites on their membranes. These findings are related to quantitative data on the concentration of peptides in the human carotid body from a previous paper with which we were associated.     

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog,Rana catesbeiana

Summary The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).     

Biogenic Amine-Stimulated Cyclic Adenosine-3'',5''-Monophosphate Formation in the Rat Carotid Body

The subcutaneous injection of isoprenaline, salbutamol, histamine, and adrenaline to rats, which were subsequently killed by microwave irradiation, resulted in a rapid increase in the cyclic AMP content of the carotid body. On the other hand, noradrenaline, dopamine, adenosine, and 5-hydroxytryptamine, at doses at least 100 times greater than that of isoprenaline, did not significantly alter the cyclic nucleotide content in vivo. The response to isoprenaline was dose related, with an ED50 of 15 micrograms X kg-1, and reached a peak level 1-1.5 min after injection. Incubation of intact carotid bodies with isoprenaline (10(-5) M) in vitro also resulted in a 10-fold increase in cyclic AMP content. The in vivo response to isoprenaline could be blocked stereo-selectively by propranolol, and ICI 118.551, a beta 2-selective antagonist, blocks the isoprenaline-elicited increase in cyclic AMP completely at a dose of 30 micrograms X kg-1; whereas betaxolol, a beta 1-selective antagonist, was ineffective, even at a dose of 300 micrograms X kg-1. Hypoxia (5% oxygen in 95% N2) did not result in a significant increase in the cyclic AMP content, nor did it significantly alter the isoprenaline-stimulated increase in the cyclic AMP content of the rat carotid body. These results suggest that some catecholamines may stimulate cyclic AMP formation by interacting with a beta 2-adrenoceptor in the rat carotid body.     

Responses of non-spiking giant interneurons to substrate tilt in the crayfish, with special reference to multisensory control in the compensatory eyestalk movement system

In the brain of the intact crayfish, three pairs of non-spiking giant interneurons (G1, G2, G3; NGIs) scarcely responded to substrate tilt about the longitudinal axis of the body either in the dark or in the presence of an overhead light. However, when the statolith was removed, these NGIs responded with depolarizing and hyperpolarizing potentials respectively to upward movements of the ipsilateral legs (2nd–5th pereiopods) and upward movements of the contralateral legs produced by substrate tilt. The relationships between the polarity of the potential and the direction of movement in the contralateral legs were opposite to those in the ipsilateral legs. The amplitude of the responses was proportional to the frequency (0.5-0.05 Hz) and amplitude of tilting. When the legs were moved unilaterally, the NGIs responded with depolarizing and hyperpolarizing potentials to upward movements of the ipsilateral legs and to upward movements of the contralateral legs, respectively. When the legs were moved bilaterally in the same direction by upward or downward movement of the substrate, the NGIs scarcely responded to the leg movements. A hypothetical model is presented to account for the pathways of sensory inputs to the NGIs and the role of NGIs in compensatory oculomotor system.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Localization of cholecystokinin-like and calcitonin-like peptides in infant carotid bodies: a light- and electron-microscopic immunohistochemical study

Previous immunohistochemical studies have identified several regulatory peptides in the carotid body chief cells in both humans and animals. These peptides, together with amines, may be important in the modulation of the chemoreflex by the carotid body. We report the localization and distribution of calcitonin and cholecystokinin-like (CCK) immunoreactivity in chief cells of human infant carotid body by light- and electron-microscopic immunohistochemical techniques. Consecutive sections immunostained with calcitonin and/or CCK antibodies revealed positively stained chief cells, both alone and in clusters, scattered throughout the carotid body lobule. Generally more chief cells were positive for calcitonin than for CCK. This was confirmed by quantiative analysis showing that the ratio of calcitonin to CCK immunoreactive cells was consistently >2:1 in all cases studied. There was no apparent correlation between the immunoreactivity for the two peptides and the age, sex, or postmortem interval. Calcitonin-like and CCK-like immunoreactivities were localized electron-microscopically over the dense core granules of the chief cells. Calcitonin and CCK-like peptides in carotid body chief cells may act as neutransmitters or neuromodulators involved in chemoreception.     

Effects of intracerebroventricular administration of aminophylline on hyperbaric-induced increase in carotid blood flow

Carotid blood flow was measured in rats by implanted transit-time ultrasonic flowprobes during hyperbaric experiments at up to 70 bar (7 MPa) using an helium-oxygen hyperoxic (partial pressure of O₂ = 400 mbar) mixture. Before the hyperbaric experiment, an intracerebroventricular injection of phosphate saline buffered solution (PBS) or aminophylline, an adenosine receptor blocker, in PBS was given. Throughout the hyperbaric experiment carotid blood flow increased with ambient pressure in both PBS, i.e. control, and aminophylline treated rats. The increase in carotid blood flow was significantly attenuated in aminophylline treated rats. Additional experiments showed that the increased carotid blood flow was independent of hyperoxia as well as of temperature. The hypothesis that the hyperbaric dependent increase in carotid blood flow was mediated by brain adenosine receptors and its implication regarding a cerebral vasodilatation are discussed.