Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rosetting of human T lymphocytes with sheep and human erythrocytes. Comparison of human and sheep ligand binding using purified E receptor

Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes.     

Modulation of macrophage C3b receptor function by cytochalasin-sensitive structures

A quantitative rosette assay was employed in order to determine if through pharmacologic probes we could gain an insight into the nature of the interaction between C3b-coated particles and the macrophage C3b receptor. Rabbit alveolar macrophage monolayers were challenged with chromium-labeled, complement-coated (via cold agglutinin) human erythrocytes (HEC3b) and the per cent of bound counts determined in the distilled water lysate. With this assay system in which ingestion is negligible, the cytochalasins (A greater than E greater than D greater than B) produced the most marked inhibition of rosette formation compared to control treated monolayers. No agent examined produced consistent augmentation. Cytochalasin A at 10(-5), 10(-6), and 10(-7) M inhibited rosette formation by 77+/- 2, 44 +/- 4 and 15 +/- 7 (S.E.), per cent, respectively. Cytochalasin E was also markedly inhibitory, Cytochalasins B and D produced approximately 30% inhibition at 10(-5) M. The cytochalasin effect was not secondary to an interaction between these agents and complement-sensitized erythrocytes, although cytochalasin E was also able to reduce erythrocyte-bouund C3b reactivity. Cytochalasin A and E modulation of the macrophage C3b reactivity occurred within a few minutes and was only slightly reversible. Cytochalasins A and E could also disrupt performed rosettes but the effect was not as pronounced as when these agents were present before and/or during the actual adherence phenomenon. Vinblastine and colchicine (10(-5) and 10(-6) M) also produced significant inhibition of rosette formation, although the magnitude of the effect was less than that for cytochalasins A and E. Further characterization of the vinblastine and colchicine effect demonstrated that the inhibition was rapid, irreversible over a 60-min incubation, and not explained by an alteration in macrophage attachment or in HEC3b reactivity. Agents producing insignificant inhibition of rosette formation included the following: dibutyryl cAMP and cAMP agonists (PGE1, theophylline), 8-bromo cGMP and cGMP agonists (carbachol, asorbic acid), dimethylsulfoxide, heparin, ethanol, dextran sulfate, DEAE-dextran, and poly-L-lysine. The data suggest that cytochalasin, vinblastine and colchicine sensitive membrane structures, most likely microfilaments and microtubules, are important in the interaction of C3b-coated particles with the macrophage C3b receptor.     

人和猴T淋巴细胞表面TRBC受体和E受体的比例研究

In 1985, rosette formation of human and macaque pan-T lymphocytes with tree shrew red blood cells (TRBC) (TRBC rosette) was first found by Ben K et al, showing different physico-chemical properties from that of rosette formation with sheep red blood cells (E-rosette). In order to approach the correlation between TRBC receptor, E receptor (CD2) and other differentiation antigens (CDs) on T lymphocytes, rosette inhibition assay and antigenic modulation or co-modulation were performed with monoclonal antibodies (McAbs) to CDs, and the distribution of TRBC receptor in other peripheral immunocytes, cell lines was also examined. TRBC rosette appeared in 88.8% of E rosette positive peripheral blood lymphocytes (E(+)-PBL) and in 4.16% of E(-)-PBL. TRBC receptor was also found on all T cell lines tested (CEM, H33 HJ-JA 1, Jurkat, MLA-144, Molt-3, Molt-4, Molt-4 clone 8, PEER) and some myeloid lines (U 937 and HL 60), but not on human granulocytes, B cell lines (Daudi, Raji and Reh) and myeloid line K 562. The modulation or co-modulation of CD 3, TCR, CD 5, CD 6 and CD 7 with McAbs OKT 3, T 108 (F 1), T 136 (F 101-15), T 149 (M-T 604) and T 152 (7 G 5) did not affect TRBC rosette formation of PBL. TRBC rosette of human and rhesus monkey PBL was not inhibited by T 11.1 McAb OKT 11 (CD 2 McAb), in contrast human and rhesus monkey E rosette formations were obviously blocked at inhibition rates of 77.9% and 49.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in oxygen consumption and cyclic AMP formation of rat cerebral cortex slices

Effects of TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L- prolinamide ) and pentobarbital, alone or in combination, on oxygen consumption and cyclic AMP formation in rat cerebral cortex slices were investigated. The oxygen consumption of rat cerebral cortex slices as measured with a Warburg apparatus, increased linearly over time (0 to 60-min incubation at 37C). Addition of pentobarbital (1 to 7 x 10-4M) inhibited oxygen consumption, in a concentration-dependent manner, up to 45% of control. A concomitant application of DN-1417 (10-5M) or TRH (10-4M) and pentobarbital (5 x 10-4M) led to a partial recovery of the pentobarbital effect. The similar anti-pentobarbital effects were observed with the addition of carbachol (10-4M) or dibutyryl cyclic AMP (10-3M), but not norepinephrine (10-4M) or dopamine (10-4M). DN-1417, TRH, carbachol, norepinephrine or dopamine at 10-4M stimulated cyclic AMP formation in the cerebral cortex slices. Addition of pentobarbital (1 to 7 x 10-4M) inhibited the cyclic AMP formation, in a concentration-dependent manner. DN-1417, TRH or carbachol at 10-4M but not norepinephrine or dopamine at 10-4M significantly reversed the reduction of cyclic AMP formation induced by pentobarbital (5 x 10-4M). Atropine (10-4M) almost completely abolished DN-1417-, TRH- and carbachol-induced cyclic AMP formation in the presence and absence of pentobarbital.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Chronic Exposure to Pertussis Toxin Alters Muscarinic Receptor-Mediated Regulation of Cyclic AMP Metabolism in Neuroblastoma × Glioma NG108–15 Hybrid Cells

Chronic pertussis toxin treatment (5 days) of NG108-15 neuroblastoma X glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10(-4)M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5-day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1-stimulated cyclic AMP production. Chronic exposure (5 days) of NG108-15 cells to carbachol (10(-5)M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24-h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.     

Human monocyte-lymphocyte interaction: a new technique.

A rosette-type assay of the physical interaction between lymphocytes and monocytes after treatment with neuraminidase-galactose oxidase (NGAO) is reported. Monocyte-lymphocyte (ML) rosette formation and subsequent lymphocyte proliferation occurred when either lymphocytes or homologous monocytes were treated with NGAO and cultured together. Maximal ML rosette formation took place at 37 degrees C 4 hr after culture in media containing 10% serum at lymphocyte to monocyte ratios of 10:1 to 20:1. The percentage of rosette formation correlated with the extent of thymidine incorporation when increasing concentrations of NGAO were used. When NGAO-treated monocytes were added to untreated T and non-T lymphocytes, they bound preferentially to T lymphocytes and induced proliferation only in the T subpopulation. These results indicate that the ML rosette assay measures a highly specific monocyte-lymphocyte physical interaction after a mitogenic stimulus which is an early event in lymphocyte activation since it reflects the degree of subsequent lymphocyte proliferation.     

Modulation of the adherence of human lymphocytes to measles-infected cells by prostaglandin E1: A differential effect on lymphocyte subpopulations

Prostaglandins of the E series (PGE) may serve as important regulators of human immune responsiveness. The present study was designed to examine the possibility that PGE may effect human lymphocyte function by the modulation of surface receptors. The presence of surface binding sites on human lymphocytes for measles virus antigens was studied using a rosette adherence assay. We observed that the addition of PGE₁ increased the proportion of measles-infected cells (Hela-Kll) with adherent lymphocytes (75% increase at 3 × 10⁻⁶ M PGE₁). PGE was observed to enhance the adherence of purified normal peripheral T cells (87%) and T lymphoid cells (Molt 3) (27%). In contrast, no significant change in normal peripheral B cell or B lymphoid cell (Raji) adherence was observed with the addition of PGE. These results are consistent with a selective modulation of surface measles virus binding sites by PGE₁ on T and not B lymphoid cells.     

Specific Inhibition of Dopamine D-1-Mediated Cyclic AMP Formation by Dopamine D-2, Muscarinic Cholinergic, and Opiate Receptor Stimulation in Rat Striatal Slices

The ability of different receptors to mediate inhibition of cyclic AMP accumulation due to a variety of agonists was examined in rat striatal slices. In the presence of 1 mM 3-isobutyl-1-methylxanthine, dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10(-8)-10(-5) M), respectively, inhibited the increase in cyclic AMP accumulation in slices of rat striatum due to dopamine D-1 receptor stimulation by 1 microM SKF 38393. In contrast, these inhibitory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin E1, 2-chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in striatal slices. These results suggest that in rat striatum either dopamine D-2, muscarinic cholinergic, and opiate receptors are only functionally linked to dopamine D-1 receptors or that the D-1 and D-2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels.     

Potential mediation of prostaglandin E2 release from isolated human parietal cells by protein kinase C

Parietal cells are a major source of gastric mucosal prostaglandins in various species. We examined cholinergic stimulation of prostaglandin E2 (PGE2) release from human parietal cells; using activators of the protein kinase C we attempted to get an indirect insight into cellular mechanisms which control PGE2 release. Gastric mucosal specimens were obtained at surgery and the cells were dispersed by collagenase and pronase E. Parietal cells were enriched to 65-80% by a Percoll gradient, and were incubated for 30 min. PGE2 release into the medium (radioimmunoassay) was 74-126 pg/10(6) cells/30 min under basal conditions and was 2.6-fold increased by carbachol (10(-5) and 10(-4) M). Similarly, PGE2 release was stimulated by phospholipase C (20-200 mU/ml, 364% above basal), 1-oleoyl-2-acetyl-sn-glycerol (10(-9)-10(-5) M, 229%), 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9)-10(-5) M, 283%) and calcium ionophore A23187 (10(-7)-10(-5) M, 219%). Simultaneous presence of A23187 and TPA synergistically induced stimulation which was slightly higher than the sum of the individual responses. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide W-7, a putative calmodulin antagonist, inhibited TPA-induced PGE2 release at concentrations regarded specific for blocking calmodulin (IC50 = 1.5 X 0(-6) M). We conclude that in human parietal cells PGE2 is released upon cholinergic stimulation and that phospholipase C and protein kinase C are involved in the control of PGE2 release. We speculate that calmodulin might interact with a protein phosphorylated by protein kinase C to cause PGE2 release.     

Ketotifen increases cyclic adenosine 3',5'-monophosphate in intact human lymphocyte and potentiates other adenylate cyclase activating agents

We have investigated the effects of ketotifen on the cyclic adenosine 3',5'-monophosphate (cyclic AMP) response of intact human lymphocyte and its interaction with adenylate cyclase activating agents. In the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine), ketotifen (10(-8)-10(-4) M) caused an 80% increase in cyclic AMP content of human lymphocyte, a magnitude similar to that observed with hydrocortisone. The cyclic AMP level peaked at about 15 minutes and remained elevated for at least 45 minutes. In addition, ketotifen (10(-6)-10(-4) M) markedly potentiated the effect of several adenylate cyclase stimulating agents, including L-isoproterenol, prostaglandin E1 and forskolin. The biochemical mechanisms underlying these effects are unknown. It may be at least partly related to the ability of ketotifen to reverse and prevent beta 2 adrenoceptor desensitization and to promote the formation of hormone - nucleotide - high affinity receptor complex. These effects may contribute to its prophylactic effect in the treatment of bronchial asthma.     

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.     

Effects of cordacin on lymphocyte activity in vitro

The effects of cordacin on lymphocyte activity were studied with two parameters in vitro. The cordacin inhibited T lymphocyte E rosette formation by 93% at the concentration of 1,000 microgram/ml and tritiated-thymidine incorporation into PHA-transformed lymphocytes by 66% at the concentration of 10 microgram/ml. The implication of these results on replicating leukemic as well as normal cells is discussed.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Cholinergic-Adrenergic Receptor Interactions in Cerebral Microvessels

Enriched capillary preparations isolated from rat cerebral cortex were used to evaluate cholinergic-adrenergic receptor interactions in cerebral endothelium. Possible receptor interactions were determined by measuring an intracellular mediator, cyclic AMP and alterations in GTP-sensitive agonist binding. Unstimulated microvessel homogenates generate 66 +/- 16 pmol/mg/10 min of cyclic AMP. Adrenergic agonists norepinephrine and isoproterenol increase cyclic AMP to 147 +/- 31 and 149 +/- 23 pmol/mg/10 min, respectively. Addition of the muscarinic agonist carbachol has no effect on basal cyclic AMP but it completely blocks the stimulation elicited by adrenergic agonists. The displacement of quinuclidinyl benzilate (QNB) by carbachol yields an IC50 of 1.5 +/- 0.45 X 10(-4) M and a Hill coefficient of 0.54 +/- 0.07, indicating a heterogeneous population of binding sites. Guanine nucleotides shift the displacement curve to the right (IC50, 4.7 +/- 0.16 X 10(-4) M) and convert the binding site population to greater homogeneity (0.76 +/- 0.18). Isoproterenol prevents both the affinity shift and binding site conversion evoked by guanine nucleotides. These data suggest that cholinergic-adrenergic interactions occur at both the level of receptor binding and the generation of an intracellular messenger. Since cyclic AMP has been purported to play a role in regulation of blood-brain barrier permeability, the existence of adrenergic-cholinergic, i.e., excitatory-inhibitory modulators of adenylate cyclase in cerebral endothelium, suggests that these receptors may mediate physiological and/or pathological alterations of cerebrovascular permeability.     

Metabolic and morphologic effects of colchicine on human T-lymphocyte expression of Fc mu and Fc gamma receptors

The influence of colchicine on human T-cell Fc mu- and Fc gamma-receptor expression during culture was studied utilizing a rosette technique with bovine erythrocytes coated with IgM (EOx-IgM) or IgG (EOx-IgG). Treatment of T cells with greater than or equal to 10(-6) M concentrations of colchicine induced in these cells progressive loss of microtubules and surface microvilli, inhibited their Fc mu-, but not Fc gamma-receptor expression during culture, and increased their cyclic AMP levels. However, similar treatment of cells with lumicolchicine, a photoinactivated isomer, identically inhibited the T-cell Fc mu-receptor expression as well, without inducing loss of microtubules or microvilli or raising cyclic AMP levels in them. A direct influence on T-cell protein synthesis by either colchicine or lumicolchicine is likely, as greater than or equal to 10(-6) M concentrations of alkaloid identically inhibited [3H]leucine incorporation and Fc mu-receptor expression by T cells without inhibiting their alpha-methyl isobutyric acid transport. No impairment of optimal EOx-IgM rosette formation occurred in control T lymphocytes cultured for 24 hr and then treated with colchicine, which suggests that its effects did not directly influence the receptor-ligand interaction itself. These findings suggest colchicine has several sites of action on T cells, dependent and independent of microtubular depolymerization, which may be responsible for alterations of T-lymphocyte cellular metabolism and function.     

Serotonin-activated adenylate cyclase and the possible role of cyclic AMP in modulation of buccal muscle contraction in Aplysia

The possible role of cyclic AMP in mediating opposite modulatory effects of serotonin (5-HT) on Aplysia buccal mass muscles E1 and E2 was examined. Serotonin enhances E1 contractions and inhibits E2 contractions. Adenylate cyclase in membranes of both E1 and E2 is stimulated approximately 180% by 10(-6) M 5-HT and 300% by 10(-3) M 5-HT. Dibutyryl cyclic AMP and 8-benzylthio cyclic AMP mimicked the effect of 5-HT on E1 but had no effect on E2. Theophylline (Th) and isobutylmethylxanthine (IBMX) mimicked the effect of 5-HT on E1 at high concentrations. Concentrations of Th and IBMX low enough not to have any direct effect on contraction increased both the magnitude and duration of the effect of 5-HT on E1 contraction. Neither Th nor IBMX had a direct effect on E2 contraction, although Th produced a small increase in the effect of 5-HT on E2. These data are consistent with the hypothesis that cyclic AMP mediates the enhancement effect of 5-HT on E1 contraction. Other mechanisms probably mediate the effect of 5-HT on E2 contraction.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.