Histamine H2 receptors on chondrocytes derived from human, canine and bovine articular cartilage.

Histamine (1-100 microM) induced a concentration-dependent increase in intracellular cyclic AMP in monolayer cultures of human, canine and foetal-bovine articular chondrocytes. The dose-response curve for histamine in each culture was progressively displaced to the right with increasing concentrations of cimetidine, an H2-receptor antagonist. The histamine-induced cyclic AMP elevation in human articular chondrocytes was also significantly decreased by ranitidine, another H2 antagonist, but not by the H1 antagonists mepyramine and chlorpheniramine. These findings indicate that histamine activates chondrocyte adenylate cyclase through an H2 receptor. The cyclic AMP response of human chondrocytes to histamine was many times greater than that measured for synovial fibroblasts under similar conditions. Such findings suggest that mast-cell-chondrocyte interactions in vivo may contribute to changed chondrocyte metabolism in joint disease.     

Estradiol receptors in articular chondrocytes

Recent experimental studies suggest a role for estrogens in chondrocyte metabolism and the development and treatment of osteoarthritis. Type I cytoplasmic estradiol receptors were found in articular chondrocytes. The presence of these receptors supports a possible mechanism for previous observations on estrogen suppressive effects on cartilage proteoglycan synthesis and offers a basis for further study on the role of estrogens in the physiology of cartilage and in the pathogenesis and treatment of osteoarthritis.     

Histamine H2 receptors and cyclic AMP in brain

The intravenous injection of histamine to 2–3 day old chicks resulted in a rapid and marked increase in the cyclic AMP content of the cerebral hemispheres that had been removed and frozen within 0.5s using a freeze-blowing technique. This response was not antagonized by pretreatment of the chicks with the histamine H₁-receptor antagonists mepyramine and diphenhydramine but was blocked by the H₂-receptor antagonists burimamide and metiamide. Parallel $\text{in vitro}$ experiments on slices of chick cerebral hemispheres demonstrated that the H₁ antagonists only produced a weak and non-competitive antagonism of the effects of histamine on cyclic AMP production. On the other hand the H₂ antagonists at low concentrations competitively blocked the histamine response. It is suggested that increased cyclic AMP formation in chick cerebral hemispheres can be mediated through stimulation of histamine H₂-receptors.     

Histamine excites rat cerebellar granule cells in vitro through H1 and H2 receptors.

The effects of histamine on the firing of cerebellar granule cells were investigated in vitro. Histamine predominantly produced excitatory (117/123, 95.1%) and in a few cases inhibitory (6/123, 4.9%) responses in granule cells. The histamine-induced excitation was not blocked by perfusing the slice with low Ca2+/high Mg2+ medium, supporting a direct postsynaptic action of histamine. The H1 receptor antagonists triprolidine and chlorpheniramine significantly diminished the histamine-induced excitation, but the H2 receptor antagonist ranitidine did not significantly reduce the excitation. On the other hand, the H2 receptor agonist dimaprit could elicit a weak excitation of granule cells. This dimaprit-induced excitation was blocked by ranitidine but not triprolidine. These results reveal that the excitatory effect of histamine on cerebellar granule cells is mediated by both H1 and H2 receptors with a predominant contribution of H1 receptors. The relevance of these findings to the possible function of the hypothalamocerebellar histaminergic fibers in cerebellum is discussed.     

Histamine H1 and H2 receptors but not H4 receptors are upregulated during bone marrow regeneration

The role of histamine receptors in radiation-induced bone marrow (BM) regeneration was investigated with aspects of functional genomics. H1R and H2R mRNA expression increased during regeneration in both histidine decarboxylase knockout (HDC-/-) and wild type (HDC+/+) mice, though to a lesser extent in HDC-/- mice. H4R mRNA expression was downregulated in both groups. Mainly CD34+ cells were responsible for the elevation of intracellular histamine and HDC content in HDC+/+ BM cell populations. The differential changes in the expression of its receptors, and also its elevated levels in hematopoietic progenitors support the regulatory role of histamine in BM regeneration, that could be further explored by future gene expression studies.     

Identification of a novel inflammatory stimulant of chondrocytes. Early events in cell activation by bradykinin receptors on pig articular chondrocytes.

The inflammatory peptide bradykinin stimulated a rapid and transient increase in cytoplasmic [Ca2+] in primary pig chondrocytes, as measured by the fluorescent indicator dye Fura-2. This increase occurred in the absence of extracellular Ca2+, indicating a mobilization from intracellular stores. The elevation in intracellular [Ca2+] was mediated by authentic bradykinin receptors, since it was blocked by the specific bradykinin antagonist [beta-(2-thienyl)-L-Ala5,8,D-Phe7]bradykinin. Activation of chondrocytes by bradykinin induced a concentration-dependent [ED50 (dose for half-maximal response) approximately 40 nM] accumulation of inositol monophosphate in the presence of LiCl and a concentration-dependent increase in production of prostaglandin E2. The generation of the secondary mediator prostaglandin E2 was a biologically relevant output response induced by bradykinin, but chondrocyte responses, such as the rate of entry into DNA synthesis, the rate and pattern of new protein synthesis and the rate of synthesis and resorption of cartilage proteoglycan, were unaltered by bradykinin treatment. Chondrocytes were also shown to be activated by two pharmacological mediators of cytosolic [Ca2+] elevation, i.e. the ionophore A23187 and thapsigargin, which both produced alterations in protein synthesis which were mimicked by bradykinin. Thus Ca2+-sensitive pathways exist which are not functionally responsive to a Ca2+-mobilizing and inositol phosphate-generating hormone, potentially indicating other routes of regulation. These results call attention to bradykinin and related peptides as another class of inflammatory mediators which may regulate physiological and pathological chondrocyte metabolism.     

Electrophysiological demonstration of voltage- activated H+ channels in bovine articular chondrocytes.

Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pH(i)), so characterising the membrane transport pathways that determine pH(i) is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltage-activated H(+) channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H(+), the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pH(o)) and pH(i), and inhibited by Zn(2+). The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.     

Histamine H3 receptors modulate reactive hyperemia in rat gut.

Reactive hyperemia (RH) is an abrupt blood flow increase following release from mechanical occlusion of an artery, with restoration of intra-arterial pressure. The mechanism of this postocclusion increase in blood flow in the gut is multifactorial. Relaxation of intestinal resistance vessels, observed during RH, may involve myogenic, metabolic, hormonal and neurogenic factors. Evidence exists that histamine is an important endogenous mediator of various functions of the gut, including blood flow. The vascular effects of histamine in the intestinal circulation are due its agonistic action on histamine H1, H2 and H3 receptors. In the present study the hypothesis was tested that peripheral histamine H3 receptors are involved in the mediation of RH in the intestinal circulation. In anesthetized rats, anterior mesenteric artery blood flow (MBF) was determined with ultrasonic Doppler flowmeter, and arterial pressure (AP) was determined with a transducer. The increase in the volume of blood accumulating during RH (RH-volume), the peak increase of arterial blood flow (RH-peak response) and the duration of the hyperemia (RH-duration) were used to quantify RH after occluding the anterior mesenteric artery for 30, 60 and 120 s. Hyperemia parameters were determined before and after administration of the selective histamine H3 receptor antagonist clobenpropit. Pretreatment with clobenpropit was without any effect on control MBF and AP but significantly reduced most of RH responses. These findings support the hypothesis that histamine H3 receptors do not play any role in the control of intestinal vasculature at basal conditions but these receptors participate in the intestinal hyperemic reaction in response to complete temporal intestinal ischemia.     

Estrogen receptors in cultured rabbit articular chondrocytes: influence of age

Primary cultures of pubertal and prepubertal rabbit articular cartilage cells were performed. Total homogenates or cell extracts were used to determine the specific binding of 17 beta-estradiol. A comparative study was undertaken with tissue minces homogenized without enzymatic treatment. Scatchard analysis of cell or tissue extracts revealed the presence of a high-affinity receptor with Kd values of 0.55 +/- 0.16 nM and 0.12 +/- 0.03 nM in prepubertal and pubertal rabbit chondrocytes respectively. A significant difference in the affinity of estrogen receptor for its ligand as a function of age was observed. In contrast there was no significant difference in the number of binding sites expressed as fmol per mg DNA between the two age groups. The ligand binding specificity was as expected for an estrogen receptor and the sedimentation coefficient was 3.2 S when analyzed by ultracentrifugation on sucrose density gradient in presence of 0.4 M KCl and 8.1 S in low salt conditions. The binding sites, labeled with [125I]estradiol, were specifically immunoprecipitated by a monoclonal antibody to the estrogen receptor (JS34/32).     

Pharmacological actions of 17 beta-oestradiol on articular cartilage chondrocytes and chondrosarcoma chondrocytes in the absence of oestrogen receptors

(1) Pharmacological concentrations (greater than 10(-5) M) of 17 beta-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30-50%. Physiological concentrations (10(-9)-10(-8) M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10(-4) M cortisol had no effect on articular chondrocyte cell cultures. 10(-4) M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17 beta-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10(-5) M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10(-7) M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17 beta-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocytes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.     

Human articular chondrocytes acquire 1,25-(OH)2 vitamin D-3 receptors in culture

The vitamin D endocrine system is crucial in calcium homeostasis in mammalian species. Central to this role 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) receptors have been detected in freshly isolated osteoblast-like bone cells and it has been shown that the active metabolite of vitamin D-3 1,25-(OH)2D3, increases bone resorption in vitro and in vivo. The requirement of 1,25-(OH)2D3 for the normal development of growth plate cartilage can be seen in vitamin D deficient rickets. However, there is still considerable controversy regarding the presence of 1,25-(OH)2D3 receptors in chondrocytes. In this paper, we report the presence of a 3.5-S 1,25-(OH)2D3-binding macromolecule in freshly isolated human costal but not articular chondrocytes. After subculture, both articular and costal chondrocytes have receptors. Saturation binding analysis revealed a single class of binding sites with an apparent Kd of 0.09 nM and approx. 2700 receptor molecules per cell for articular chondrocytes and a Kd of 0.1 nM and approx. 2000 receptor molecules per cell for costal chondrocytes. The presence of 1,25-(OH)2D3 receptors did not correlate with the switch from synthesis of cartilage-specific type II collagen to types I and III collagens. The acquisition of 1,25-(OH)2D3 receptors by articular chondrocytes may, therefore, be another phenotypic characteristic of cultured cells or may appear in vivo when chondrocytes are exposed to vascular or inflammatory cell products.     

Histamine and H1 -histamine receptors faster venous circulation

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H(1) -histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H(1) -histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H(1) -histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.     

Histamine and 5-hydroxytryptamine-inhibition of transmitter release mediated by H2- and 5-hydroxytryptamine receptors.

The vasodilatation caused by histamine and 5-hydroxytryptamine may be due, at least in part, to their inhibitory action on adrenergic neurotransmission. The evidence for this is as follows: 1) contractions of isolated canine vascular strips caused by sympathetic nerve stimulation are depressed by these substances whereas contractions caused by norepinephrine are either unchanged or augmented; 2) histamine and 5-hydroxytryptamine inhibit the release of norepinephrine evoked by sympathetic nerve stimulation of isolated vascular strips previously incubated with the labeled transmitter. This inhibitory effect can be demonstrated using concentrations of the substinces less than those required to affect the smooth muscle cells directly. By contrast, neither histamine nor 5-hydroxytryptamine inhibits the displacement of neuronal norepinephrine by tyramine, suggesting that these substances interfere with the exocytotic process. Additional studies have identified the histamine-H2 receptor as the probable mediator of this prejunctional action of histamine, whereas the nature of the receptor for 5-hydroxytryptamine remains to be clarified.     

Modulation of H+ transport mechanisms by interleukin-1 in isolated bovine articular chondrocytes.

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18 h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.     

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)