Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia.

Immune cell-derived opioid peptides can activate opioid receptors on peripheral sensory nerves to inhibit inflammatory pain. The intrinsic mechanisms triggering this neuroimmune interaction are unknown. This study investigates the involvement of endogenous corticotropin-releasing factor (CRF) and interleukin-1beta (IL-1). A specific stress paradigm, cold water swim (CWS), produces potent opioid receptor-specific antinociception in inflamed paws of rats. This effect is dose-dependently attenuated by intraplantar but not by intravenous alpha-helical CRF. IL-1 receptor antagonist is ineffective. Similarly, local injection of antiserum against CRF, but not to IL-1, dose-dependently reverses this effect. Intravenous anti-CRF is only inhibitory at 10(4)-fold higher concentrations and intravenous CRF does not produce analgesia. Pretreatment of inflamed paws with an 18-mer 3'-3'-end inverted CRF-antisense oligodeoxynucleotide abolishes CWS-induced antinociception. The same treatment significantly reduces the amount of CRF extracted from inflamed paws and the number of CRF-immunostained cells without affecting gross inflammatory signs. A mismatch oligodeoxynucleotide alters neither the CWS effect nor CRF immunoreactivity. These findings identify locally expressed CRF as the predominant agent to trigger opioid release within inflamed tissue. Endogenous IL-1, circulating CRF or antiinflammatory effects, are not involved. Thus, an intact immune system plays an essential role in pain control, which is important for the understanding of pain in immunosuppressed patients with cancer or AIDS.

Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation.

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.

Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects.

Epidermal Langerhans cells (LC) are associated anatomically with epidermal nerves, and a product of these nerves, calcitonin gene-related peptide (CGRP), inhibits the antigen-presenting capacity of LC and macrophages. As the CGRP receptor appears to be coupled to Gs alpha protein, which in turn activates adenylate cyclase, the ability of CGRP to induce cAMP in LC was examined and correlated with functional effects. LC were isolated from murine epidermal cells using antibodies on magnetic microspheres. Exposure to CGRP induced a significant increase in cAMP content, which could be inhibited by coculture with a truncated form of CGRP [CGRP-(8-37)] that is a specific competitive inhibitor of CGRP. Substance P and calcitonin failed to induce cAMP in LC. Although culture in CGRP reduced the ability of murine epidermal cells enriched for LC content to present pigeon cytochrome c to a responsive clone or to present antigen for elicitation of delayed-type hypersensitivity in immune mice, culture in forskolin had little or no effect on antigen presentation despite increased cAMP content of LC as much or more than that induced by CGRP. The effect of CGRP on antigen presentation in these systems could be blocked with CGRP-(8-37). CGRP inhibited the induction of B7-2 by lipopolysaccharide on peritoneal macrophages and a LC line, whereas calcitonin did not. CGRP induces specific accumulation of cAMP in LC and inhibits LC antigen-presenting function by a receptor-mediated event. However, the induction of cAMP by itself does not account for inhibition of antigen presentation. Suppression of the expression of B7-2 may be one mechanism by which CGRP inhibits antigen presentation.