β1-and β2-adrenoceptor-mediated secretion of amylase from incubated rat parotid gland

The present in vitro investigation was undertaken in an attempt to obtain further information on β-adrenoceptor specificity and action in the rat parotid gland, with regard to amylase secretion. The β₁-selective agonist prenalterol was roughly 800 times more potent than the β₂-agonist terbutaline, and about 5 times more effective than noradrenaline in evoking amylase release. Propranolol was the most effective inhibitor of amylase release in all experiments. The β₁-selective antagonist metoprolol and H104/08 were also effective blockers of maximal noradrenaline-and prenalterol-induced release. The inhibition curves displayed biphasic shapes when amylase secretion was induced by noradrenaline, but not when prenalterol was the secretagogue. The β₂-antagonist H35/25 was without effect on maximal noradrenaline-and prenalterol-stimulated secretion. The amylase release evoked by submaximal concentration of terbutaline was inhibited by the two antagonists H35/25 and IPS 339. In another series of experiments propranolol and metoprolol clearly shifted the noradrenaline concentration-response curve to the right, whereas H35/25 was without effect. The results further demonstrate the major importance of the β₁-adrenoceptor (noradrenaline-activated) in eliciting amylase release from the rat parotid gland. However, it is also suggested that the β₂-adrenoceptors (terbutaline-activated) may to some extent serve the same function.     

Comparison of β-adrenoceptors mediating vasodilatation in canine subcutaneous adipose tissue and skeletal muscle

Blood flow changes in response to various drugs in simultaneously autoperfused canine subcutaneous adipose tissue and gracilis muscle were compared to study the vascular β-adrenoceptors. Compared to isoprenaline the β₂-selective agonist salbutamol was 4–6 times more potent as a vasodilator in the muscle than in adipose tissue. Furthermore two β₁-selective agonists (Tazolol and H80/62) caused vasodilatation in adipose tissue but not in the gracilis muscle. When given by close i.a. injection after β-adrenoceptor blockade, adrenaline was a more potent vasoconstrictor than noradrenaline in both tissues. Before β-blockade, however, noradrenaline was the more potent vasoconstrictor in the gracilis muscle whereas adrenaline was more potent in adipose tissue. Intravenous infusion of adrenaline in doses causing vasodilatation in the muscle caused vasoconstriction in adipose tissue whereas intravenous infusion of noradrenaline caused vasoconstriction in both tissues. The present findings suggest that the β-adrenoceptors mediating vasodilatation in skeletal muscle are mainly of the β₂-type, whereas β₁-adrenoceptors seem to predominate in subcutaneous adipose tissue. Since adrenaline is a much more potent β₂- than β₁-agonist, these differences point to different roles of intravascular adrenaline in the two sites. In skeletal muscle circulating adrenaline is mainly a vasodilator whereas in subcutaneous adipose tissue it mainly acts as a vasoconstrictor.     

Effects of β-adrenergic agonists in the parotid gland of the rat–an electrophysiological study

The effects of some β-adrenergic agonists were studied in the parotid gland of the rat by electrophysiological techniques. In the unoperated gland, isoprenaline caused depolarizations which were slowly developing, long-lasting and of low amplitude. The same response was seen when noradrenaline was combined with α-adrenoceptor blocking drugs. A greater number of cells responded to this combination than to isoprenaline. After either parasympathetic or sympathetic denervation 1–3 weeks in advance, to induce supersensitivity, the number of cells responding to β-adrenoceptor stimulating drugs was significantly increased. In the latter case the threshold dose required to evoke a response was also significantly lowered. Atropine did not have any effect on the isoprenaline-evoked response. The combined parasympathetic and sympathetic denervation did not further increase the responsiveness. It is concluded that β-adrenoceptor stimulation in the parotid gland of the rat may cause membrane depolarizations. The response is mediated by β₁- adrenoceptors. The responsiveness is increased in the denervated gland. Secretory studies have demonstrated a supersensitivity to β-adrenergic agonists as a result of denervation. On the other hand, β-adrenoceptor stimulation is believed mainly to activate the adenylate cyclase/cyclic AMP system independent of membrane potential changes. It is thus not known if the present ‘supersensitivity’ is correlated to the increased secretory response earlier demonstrated in this gland.     

The importance of an intact sympathetic innervation for the differentiation of the β-adrenoceptor subtypes in the rat parotid gland

Acinar cells of the rat parotid gland are in intimate contact with adrenergic nerves, arousing from the ganglion cervicale superior (Bloom et al. 1977). The sympathetic nerves or circulating catecholamines exert their effects by activating α-adrenoceptors (electrolyte and water secretion) or β-adrenoceptors (enzyme secretion) (Batzri et al. 1971, Carlsöö et al. 1981). It is now well established that the β-adrenoceptors can be divided in two subclasses, β₁ and β₂, present in different proportions depending on the tissue examined (Lands et al. 1967, Carlsson et al. 1972, Barnett et al. 1978, Minneman et al. 1979a). The concentrations of the two subtypes are regulated independently (Minneman et al. 1979b) and their molecular structure seems to be different (Venter et al. 1981). The β₁, -subtype is dominating in normal rat parotid glands (Carlsöö et al. 1981, Ludford & Talamo 1980). Supersensitivity occuring in salivary glands after interruption of the sympathetic nerve supply in adult animals is usually associated with an increased number of the β-adrenoceptors (Ludford & Talamo 1980, Stefano & Perec 1981). In the present work we have studied the effect of neonatal sympathetic denervation of the rat parotid gland on the binding of the β-adrenoceptor radioligand ³H-dihydroalprenolol (³H-DHA) and on the β-adrenoceptor-induced amylase secretion. The results suggest that the normal dominance of the β₁-adrenoceptors is lost after neonatal sympathetic denervation. Hence, an intact sympathetic nervous system may be of major importance during the development of the β-adrenoceptor population.     

Effects of α- and β-adrenoceptor stimulation on 45Ca2+ efflux and insulin secretion from perfused rat islets

Exposure to the β₂-adrenoceptor agonist terbutaline resulted in a transient stimulation of ⁴⁵Ca²⁺ efflux from ⁴⁵Ca²⁺ preloaded rat islets perfused in 2 mm Ca²⁺ and 8.3 mm glucose. Concomitantly, an increase in insulin secretion occurred. Under the same experimental conditions, the α-adrenoceptor agonist noradrenaline promptly inhibited insulin release without any apparent influence on ⁴⁵Ca²⁺ efflux. In contrast, in a medium containing 2 mm Ca²⁺ and a low glucose concentration (2.8 mm), terbutaline stimulated insulin secretion without any apparent effects on ⁴⁵Ca²⁺ efflux. Noradrenaline had no effect on insulin secretion or ⁴⁵Ca²⁺ efflux in this medium. When islets were perfused with 8.3 mm glucose in a Ca²⁺ deficient medium, with or without addition of the chelating agent EGTA, terbutaline induced a marginal stimulation of insulin secretion and a negligible stimulation of ⁴⁵Ca²⁺ efflux. On the contrary, noradrenaline stimulated to an immediate and notable ⁴⁵Ca²⁺ efflux in these Ca²⁺ deficient media. Noradrenaline also clearly inhibited insulin secretion, though less markedly and with a slower onset than in islets perfused in 2 mm Ca²⁺. When the islets were perfused in a Ca²⁺ deficient medium with 2.8 mm glucose, terbutaline had a slight insulin releasing effect, but stimulated ⁴⁵Ca²⁺ efflux potently. Noradrenaline had no influence on insulin secretion but a weak stimulatory effect on ⁴⁵Ca²⁺ efflux. The data suggest that the β₂-adrenoceptor agonist terbutaline has the ability to stimulate insulin secretion in perfused rat islets, requiring extracellular Ca²⁺ for the full expression of its effects. These effects may be exerted through a Ca²⁺-Ca²⁺ exchange over the cell membrane and/or through cAMP and intracellular Ca²⁺ perturbations. Moreover, terbutaline directly stimulates ⁴⁵Ca²⁺ efflux, an effect inhibited by glucose. Further, the α-adrenoceptor agonist noradrenaline can inhibit insulin secretion in the absence of extracellular Ca²⁺, but the full expression of its inhibitory action is dependent on extracellular Ca²⁺ and glucose. In addition, noradrenaline stimulates ⁴⁵Ca²⁺ efflux in a Ca²⁺ deficient medium, an effect which appears independent of the glucose concentration.     

MAdCAM-1 costimulates T cell proliferation exclusively through integrin α4β7, whereas VCAM-1 and CS-1 peptide use α4β1: evidence for “remote” costimulation and induction of hyperresponsiveness to B7 molecules

We have analyzed the effects of the α⁴ integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the α⁴ β₁ and α⁴ β₇ integrins, medicate T cell costimulation exclusively through integrin α⁴ β₁, but not through α⁴ β₇. The inability of VCAM-1-Fc to costimulate via α⁴ β₇ suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via α⁴ β₇, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody (“remote” costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc > ICAM-1-Fc > MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

αEβ7 and α4β7 integrins associated with intraepithelial and mucosal homing,are expressed on macrophages

The two β₇ integrins α_Eβ₇ and α₄β₇ are the most recently described members of the integrins participating in intercellular binding. Their expression has been shown to be restricted to leukocytes and they have been suggested to be predominantly found in lymphocytes associating with the epithelium. Expression of β₇ has mainly been studied on lymphocytes whereas macrophages have been reported not to express the β₇ integrins. In this paper we have studied the expression of β₇ integrins in monocytoid cells. The myelomonocytic cell lines HL-60 and THP-1 did not express β₇ mRNA or protein, but differentiation of these cell lines to macrophages with phorbol 12-myristate 13-acetate (PMA) led to a strong induction of the β₇ mRNA expression. A clear but less pronounced up-regulation of β₇ mRNA-expression was also seen after treatment of HL-60 and THP-1 cells with interferon-γ (IFN-γ). However, its up-regulating effect on the surface expression of α₄β₇ and α_E7 complexes (detected by the monoclonal antibodies Act I and HML-1, respectively) exceeded that observed with PMA. To verify the in vitro cell line observations with normal cells, we also studied peripheral blood monocytes and tissue macrophages. Peripheral blood monocytes were Act I^? and HML-1^? in flow cytometry, but their expression was increased after a 72-h culture in the presence of PMA or IFN-γ. Also, several Act I⁺ and HML-1⁺ macrophages were found in immunohistochemical stainings of both liver and edemic lung biopsies as well as in lymph node sinuses. We therefore conclude that while monocytes do not express β₇ integrins the more differentiated cells of the monocyte-macrophage lineage do express both the α₄β₇ and α_Eβ₇ integrins, which might play a role in their intraepithelial homing.     

Influence of extracellular calcium and nifedipine on α1-and α2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca²⁺ and nifedipine on contractile responses to 10 μM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly α₂-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly α₁-adrenoceptors) and CMA (sensitive to both at,- and α₂-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca²⁺-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K⁺ was more sensitive to Ca²⁺ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca²⁺, can also release Ca²⁺ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, α₁-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca²⁺ influx and intracellular Ca²⁺ release, whereas α₂-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca²⁺ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca²⁺-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca²⁺, whereas in the other types of artery, Ca²⁺ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca²⁺ influx and intracellular Ca²⁺ release to the contractile activation, but also in the nifedipine sensitivity of the Ca²⁺ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of α-adrenoceptor stimulated by NA and to the type of vessel studied.     

Effects of nimodipine,Bay K 8644 and pinacidil on α1- and α2-adrenoceptor-mediated vasoconstriction in human hand veins

The effects of nimodipine, Bay K 8644 and pinacidil, three drugs interfering with transmembrane Ca²⁺ fluxes in different ways, were investigated in isolated human hand veins. Their ability to influence the concentration-response relationship for noradrenaline (NA) was assessed in the absence and presence of prazosin or rauwolscine. The contractile response to NA was almost abolished in Ca²⁺-free medium. Nimodipine and pinacidil depressed the NA concentration-response curve both in the absence and presence of α-adrenoceptor blockers. The NA response was only partially inhibited by nimodipine, indicating that NA may activate nimodipine-insensitive influx pathways, presumably receptor-operated calcium channels. Pinacidil inhibited the contractile response to 124 mM K⁺ and reduced the NA-induced contraction in the presence of nimodipine, suggesting that pinacidil has actions other than the opening of potassium channels and subsequent membrane hyperpolarization. Bay K 8644 increased the NA potency fourfold in the presence of rauwolscine, whereas it had no effect on the NA response in the presence of prazosin and in the absence of α-adrenoceptor blockade. Such an action of Bay K 8644 can be reconciled with α-adrenoceptor activation causing membrane depolarization and opening of potential-operated calcium channels. It may be concluded that both α₁- and α₂-adrenoceptor-mediated contractions in human hand veins are highly dependent on Ca²⁺ influx, although the mechanisms utilized to bring about this influx partly differ between the two receptor subtypes.     

Identification of deletion and triple α-globin gene haplotypes in the montreal β-thalassemia screening program: Implications for genetic medicine

We obtained blood samples in a screening program designed to detect β-thalassemia heterozygotes in Montreal; additional samples were obtained from referred persons. We analyzed DNA for variant numbers of α-globin genes, notably the α-thalassemia₂ (- α/), α-thalassemia₁, (– –/), and triplicated ζ-globin gene (ααα/) haplotypes using restriction enzymes and probes for α-globin and α-globin gene sequences. We estimated the numbers of Montreal residents of Italian and Greek ethnic origin with –α/αα genotype. Thus, 4.3% of Italians and 1.5% of Greeks, or about 7,500 persons, are estimated to be α-thalassemia₂, trait (silent carriers), largely (80%) in the –α^3.7/type I form. The triplicated α-globin gene haplotype was also found. The risk of a severe (α-thalassemia₁) phenotype associated with inheritance of – –;/αα or –α/ –α genotypes was low and was found predominantly in this study, in persons of Asian ethnic origin. The sample of Asians was too small to estimate carrier frequencies; however, based on results from the β-thalassemia screening program, we estimated that about 4% of Asians (about 1,300 persons) in Montreal are α-thalassemia carriers. We identified persons heterozygous for both β-thalassemia and α-thalassemia mutations. In these double heterozygotes, the effect of the triplicated α-globin gene was to make the erythrocyte parameters used for screening (MCV and %HbA₂) more deviant from normal whereas deletion of 2 α-globin genes tended to normalize the erythrocyte values. These findings have implications for the screening program and reproductive counseling.     

Vasodilatation and modulation of vasoconstriction in canine subcutaneous adipose tissue caused by activation of β-adrenoceptors

The present experiments were undertaken to study the balance between vascular α- and β-adrenoceptors in canine subcutaneous adipose tissue during sympathetic nerve stimulation and noradrenaline injections. Propranolol potentiated and prolonged the vasoconstrictor response to close i.a. injections of noradrenaline. The vasoconstriction induced by brief nerve stimulation (0.5 to 8 Hz) was, however, unaltered by the β-adrenoceptor blockade. During prolonged nerve stimulation the vasoconstrictor response was well maintained at 1.5 Hz but at 4 Hz there was a gradual escape. The escape phenomenon at 4 Hz was diminished by propranolol. The β₁-selective antagonist practolol, like propranolol, potentiated and prolonged the vasoconstriction induced by noradrenaline injections and reduced the vasoconstrictor escape during prolonged nerve stimulation at 4 Hz. Furthermore, the vasodilatation induced by noradrenaline injection or nerve stimulation during α-adrenoceptor blockade was diminished by practolol. Practolol also blocked the lipolytic response to noradrenaiine and nerve stimulation. The β₂-selective antagonist H35/25 blocked the effects of the β₂-selective agonist salbutamol but failed to alter noradrenaline as well as nerve stimulation induced vascular and lipolytic β-adrenoceptor responses. The present results provide further support for the hypothesis that vascular β-adrenoceptors in adipose tissue are humoral (noninnervated), preferentially activated by circulating noradrenaline. Moreover, both vascular and lipolytic β-adrenoceptors activated by noradrenaline in adipose tissue are best classified as β₁-adrenoceptors.     

Modulation of oxotremorine-induced tremor by central β-adrenoceptors

The muscarinic agonist oxotremorine was used to induce tremor in rats pretreated with methylatropine. An objective assessment of tremor intensity was accomplished by means of an accelerometer-based recording system. The non-selective, lipophilic β-adrenoceptor antagonist propranolol dose-dependently suppressed tremor intensity, whereas the r -isomer of propranolol was without effect, verifying β-adrenoceptor involvement. Since the hydrophilic, non-selective β-antagonist nadolol was ineffective, the effect appears to be located inside the blood-brain barrier. The β₂-selective antagonist ICI 118, 551 dose-dependently reduced tremor intensity, whereas selective blockade of β₁-adrenoceptors with metoprolol had no effect, indicating the participation of a β₂-adrenoceptor. On the other hand, the lipophilic β₂-agonist clenbuterol dose-dependently enhanced tremor induced by oxotremorine. Determination of circulating plasma catecholamine concentrations revealed that the effect of β-antagonists on tremor was not secondary to an effect on the oxotremorine-induced rise in catecholamine levels. Thus, the results suggest that β₂-adrenocpetors located inside the blood-brain barrier are able to modulate oxotremorine-induced tremor in rats.     

Localization of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) α, β, γ in the three major salivary glands in situ of mice and their response to β‐adrenoceptor stimulation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)‐triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno‐light microscopy under non‐stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kβ was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a β‐adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno‐light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post‐exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.     

Sequence analysis of rat integrin αE1 and αE2 subunits: Tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph

The integrin α_OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human α_E2 that is predominantly expressed on intraepithelial lymphocytes. To clone α_OX-62, rat probes generated using primers specific for the human α_E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical α subunits that are closely related to but distinct from human α_E were isolated. α_E1 is predicted to be the rat homolog of mouse α_M290 and α_E2 corresponds to rat α_OX-62. Immunofluorescence analysis revealed that mouse α_E1 and rat α_E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where γδ T cells occur in lymphoid organs. Unexpectedly, α_E2 is co-expressed with intracellular CD3-δ and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.     

Selective α2-adrenoceptor activation by clonidine: effects on 45Ca2 efflux and insulin secretion from isolated rat islets

A possible role for Ca²⁺ in the α-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion was studied in isolated rat islets by the use of the selective α₂-adrenoceptor agonist clonidine. We found that clonidine, in contrast to the a,-adrenoceptor agonist phenylephrine, inhibited glucose-stimulated insulin secretion at dose levels below 10^-6 mol l^-1. In islets preloaded with ⁴⁶Ca²⁺ and perifused at 2 mmol l ^l Ca²⁺, clonidine (10^-6 moll^-1) reduced the glucose (13.3 mmol l^-1)-stimulated ⁴⁶Ca²⁺efflux during both the first and second phases of insulin secretion. Furthermore, the inhibitory effect of clonidine on glucose (13.3 mmol l^-1)-stimulated insulin secretion was partially counteracted by raising the extracellular Ca²⁺ concentrations. Moreover, the calcium channel agonist Bay K 8644 counteracted the inhibition by clonidine on glucose-stimulated insulin secretion. Our results suggest that selective α₂-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion is mediated, at least partially, by restraint of Ca²⁺-influx. This action might in turn be exerted through interference with the voltage-dependent calcium channels.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄ epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Secretory effects of 5-hydroxytryptamine following neonatal sympathectomy in rat parotid gland

Unilateral sympathetic denervation of rat parotid glands was performed within 4 h after birth. Nine weeks later the glands were used for in-vitro studies of amylase secretion, and ⁸⁶Rb+ was used as a marker for potassium efflux. The non-denervated contralateral glands served as controls. The tissue concentrations of 5-hydroxytryptamine and its metabolite 5-hydroxyindole acetic acid were also measured. 5-Hydroxytryptamine caused a significant dose-dependent increase in amylase secretion, which was inhibited by methysergide. There was no difference between controls and denervated glands. 5-Hydroxytryptamine was without effect on potassium efflux from either denervated or control glands. The sympathectomy caused increased levels of 5-hydroxytryptamine and 5-hydroxyindole acetic acid as compared with contralateral controls. The results suggest that 5-hydroxytryptamine influences the two main secretory processes in rat parotid gland differently. A significant amylase discharge was seen following 5-hydroxytryptamine stimulation, whereas no effect was seen on ⁸⁶Rb+ efflux. Although it is also proposed that there are no 5-hydroxytryptamine-associated nerves in the superior cervical ganglion innervating parotid tissue, it seems that there is a complex connection between the sympathetic pathway and the serotoninergic system.     

Distribution of α4β7 and αEβ7 integrins on thymocytes,intestinal epithelial lymphocytes and peripheral lymphocytes

β₇ is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β₇. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α₄β₇ and α_Eβ₇ using mAb against α_Eβ₇ and mAb DATK32 which recognizes a combinatorial epitope on α₄β₇. β₇⁺ thymocytes have a mature phenotype: TcR⁺, CD11a^hi CD44^hi HSA^dull. Small subsets of double-negative CD4_?CD8_?, single-positive CD4⁺ and CD8⁺ thymocytes express α₇, while double-positive CD4⁺ CD8⁺ thymocytes are β₇. However, two integrins α_Eβ₇ and α₄β₇ recognized by anti-β₇ are not expressed on an identical subpopulation of thymocytes, as β_Eα₇⁺α₄β₇^?, α_Eβ₇⁺α₄β₇⁺ and α_Eβ₇^?α₄β₇⁺ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of α_Eβ₇ but little α₄β₇. In the spleen, Peyer's patches and lymph nodes, α₄β₇ is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4⁺ and CD8⁺ lymphocytes, express high levels of β₇ in the form of α₄β₇ and α_Eβ₇, although, as observed with lymphocytes, not all α₄β₇^hi CD4^? lymphocytes expressed α₄β₇. The population of α₄β₇^hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4⁺ lymphocyte population, which can be further subdivided on the basis of α_Eβ₇, L-selectin and α₄ expression. Therefore, memory CD4⁺ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.