A histamine derivative increases intracellular calcium mobilization and oxidative metabolism in HL-60 cells.

Past work in our laboratory has shown that a derivative of histamine, histamine-trifluoromethyl-toluidide (HTMT), has surprising tissue specificity on lymphocytes and can produce remarkable immunosuppression. This study focuses on the effects of HTMT on Ca2+ mobilization and oxidative metabolism in undifferentiated and DMSO-differentiated HL-60 cells. HTMT caused two phases of increases in intracellular calcium concentrations ([Ca2+]i) in HL-60 cells. The responses were dose dependent, with similar EC50 values (1.7 x 10(-5) M for undifferentiated and 1.5 x 10(-5) M for differentiated cells). The increase in [Ca2+]i in differentiated cells was much greater than in undifferentiated cells. The maximum responses were observed after the undifferentiated cells were incubated with DMSO for 7 days. The increase in [Ca2+]i induced by HTMT in both types of cells was competitively antagonized by high concentrations of histamine but not by classic histamine receptor antagonists (H1, H2, or H3). The inhibitory effects of histamine on [Ca2+]i accumulation in differentiated cells were partially reversed by histamine H2 receptor antagonist ranitidine, whereas in undifferentiated cells, the effects of histamine on Ca2+ mobilization were not affected by ranitidine. Other cAMP elevating agents did not inhibit increases in [Ca2+]i in undifferentiated cells but did affect [Ca2+]i in differentiated cells. The enhanced response in [Ca2+]i mobilization after differentiation of HL-60 cells appeared to be the result of an increase in the expression/function of receptors for HTMT. One interesting feature of this regulation was the fact that cAMP per se did not regulate HTMT induced Ca2+ mobilization in undifferentiated cells but inhibited the mobilization in differentiated cells. HTMT caused the generation of reactive oxygen species in both undifferentiated and differentiated HL-60 cells as measured by chemoluminescence and the levels of generation correlated with the mobilization of [Ca2+]i. In addition, the EC50s for the HTMT induced calcium mobilization and the generation of reactive oxygen species were similar, as was the case for histamine induced inhibition (Ki) in both cell types. The data imply a second messenger role for Ca2+ in HTMT induced neutrophil activation.

Cyclic AMP is not a direct regulator of calcium flux and hydrolysis of phosphoinositides in human lymphocytes.

The regulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca2+ flux and phosphatidylinositol (PI) turnover in human lymphocytes were studied. cAMP did not affect the intracellular accumulation of Ca2+ induced by phytohemagglutinin (PHA) and histamine-trifluoromethyl toluidide derivative (HTMT) in peripheral blood lymphocytes (PBL). In addition, cAMP also did not alter Ca2+ flux induced by PHA, anti-CD3, or PAF in T cells, or by anti-IgM and HTMT in non-rosetted cells. Similarly, cAMP did not inhibit IP accumulation induced by HTMT in PBL, anti-CD3 in T cells, and by anti-IgM or HTMT in non-rosetted cells. The only exception was the synthesis of IP induced by PHA in T cells that was inhibited by cAMP. Furthermore, prolonged treatment of T cells with cholera toxin inhibited Ca2+ accumulation in response to CD3. The degree of inhibition of Ca2+ and IP responses was not proportional to the levels of intracellular cAMP generated.

Abnormal T suppressor cell function in juvenile rheumatoid arthritis.

The purpose of this study was to analyze T suppressor cell function in juvenile rheumatoid arthritis (JRA). JRA is a chronic inflammatory childhood disease of unknown etiology that is characterized by arthritis and immunoregulatory abnormalities. T suppressor cell precursors (CD8+, CD28-) were purified from the peripheral blood of 24 JRA patients, using a combination of monoclonal antibodies. These cells were treated with histamine or concanavalin A, agents that are known to induce suppressor activity. They were also tested for their ability to inhibit the proliferative response of autologous T cells to phytohemagglutinin. In some experiments, the accumulation of intracellular cAMP following histamine treatment was also measured. Twelve of 13 patients with clinically active JRA showed abnormal histamine-inducible T suppressor cell function, characterized by the failure of CD8+, CD28- T cells to mediate any detectable suppression. The failure of these cells to accumulate intracellular cAMP after histamine treatment was observed in 5 of 5 patients tested who had active disease. In contrast, 11 of 11 patients with clinically inactive JRA, 5 of 5 patients with cystic fibrosis, and 9 of 9 pediatric control subjects had normal histamine- and concanavalin A-inducible T suppressor cell function, and a normal cAMP response to histamine. These results suggest that patients with clinically active JRA have a reversible defect in T suppressor cell function that is associated with a failure of T suppressor cell precursors to accumulate intracellular cAMP following their exposure to selected immune stimuli.

A subset of human rosetted lymphocytes contains previously unidentified histamine.

By sequential solid-phase immunoadsorption (panning) steps, we have isolated a subset of lymphocytes (comprising 3-7% of rosetted cells) that contains high concentrations of histamine. We have used a radioenzymatic assay for the determination of histamine and have located 117 ng of histamine/1 x 10(6) cells in Leu-5+ (OKT-11), Leu-15+ cells. This subset did not contain basophils and was negative for Leu-4 (OKT-3), Leu-3 (OKT-4), Leu-2 (OKT-8), and 9.3 antigens. The function of this subset of rosetted cells has not been determined.

Histamine regulates the generation of human cytolytic T lymphocytes.

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.

In vitro pharmacologic activity of congener derivatives and model conjugates of propranolol and practolol.

Previous studies in our laboratory suggested that synthetized derivatives of isoproterenol and histamine could create agonists more potent and receptor and/or tissue selective than the parent compound. In the present study we have evaluated the hypothesis that our results with isoproterenol and histamine derivatives could be extended to include beta-adrenergic antagonists. With this purpose in mind, fourteen derivatives of propranolol and practolol were synthesized and tested in four in vitro systems. The congeners and conjugates were tested using biologic assays (blocking of cAMP accumulation) and/or radioligand binding assays in S-49 lymphoma cells and in rat adipocytes, heart and lung which contain beta 1 and/or beta 2 receptors. Our results indicate that structural modifications distant from the pharmacophore alter the pharmacologic profile of the parent compound. The relative potencies of the derivatives were dependent upon several key factors including the length of the methylene spacer chain and the nature of the substituents on the aromatic ring. The presence of a spacer group with four methylenes resulted in the most active compound in each series when tested on S-49 cells. The derivatives with a paramethyl toluidide group were more potent than the derivatives with a trifluoromethyl toluidide group. The dipeptide derivatives were more potent on adipocyte than S-49 cells, suggesting a preference for beta 1 receptors. Some of the same modifications that led to altered potency and which resulted in an increased receptor and/or tissue selectivity using the progenitors isoproterenol or histamine did extrapolate to the beta blockers. Our data suggest that alterations in receptor and/or tissue selectivity must be imparted by the carrier moiety of the drug and may be related to the biochemical microenvironment of the receptors.

Conjugates of catecholamines. VII. Synthesis and beta-adrenergic activity of peptide catecholamine conjugates.

A series of novel catecholamine derivatives has been prepared in which one of the N-methyl substituents of isoproterenol has been extended by a spacer consisting of a chain of four methylenes which terminates with an amide linkage to a peptide, the point of attachment being via the aromatic amino group of p-aminophenylalanine. In one of the derivatives, two catecholamines are attached to the same peptide in this manner. The peptides, which range in size from three to eight amino acid residues and contain phenylalanine, glycine, and L-alpha-amino-delta-hydroxyvaleric acid, were synthesized via stepwise and fragment condensation techniques. The beta-adrenergic agonist activities of the derivatives were evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells.

The effects of derivatives of histamine on natural suppressor cells.

Histamine is an impressive modulator of immune functions at least via its effects on lymphoid cells. Its in vivo effects will not be used practically as long as they produce the profound cardiovascular and pulmonary effects for which the drug is known. A series of 13 congener derivatives and conjugates of histamine was constructed and was tested to investigate whether chemical alterations would result in pharmacologic actions on leukocytes that were more potent and effect specific than histamine. The new compounds, which contained spacer groups of varying lengths between ligand and carrier and with various aromatic modifying groups, showed potencies widely different from histamine when tested in natural suppressor cells. Some compounds showed selective effects on natural suppressor cells in that they were inactive on myocardial tissue, whereas other compounds were selectively active on the myocardium. Some compounds augmented the suppressive capacity of natural suppressor cells in mixed leukocyte reactions via H1 receptors. Our scheme might be more widely extrapolated to other low m.w. immune modulators in an attempt to make them lymphocyte specific. The data also encourage the in vivo testing of selected histamine analogues as selective modulators of immunity. Some of these modulators might be experimentally useful in vivo because they may lack actions in other tissues.

Conjugates of catecholamines. 6. Synthesis and beta-adrenergic activity of N-(hydroxyalkyl)catecholamine derivatives.

A new series of catecholamines has been prepared in which the N-alkyl substituent of dl-epinephrine or dl-isoproterenol has been extended by a methylene chain terminated by a hydroxyl group or derived functionality (e.g., carbamate or ester). These functionalized catecholamines (congeners) and model compounds were prepared with the goal of eventual attachment to polymeric carrier molecules. The beta-adrenergic agonist activity of the derivatives was evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells and by the displacement of iodocyanopindolol (ICYP). A n-butylcarbamate derivative (compound 15) was the most active compound in this series with a potency 190 times greater than dl-isoproterenol in the S49 assay. The biological results indicate that minor modifications in structure in the N-alkyl substituent of the catecholamine can influence the pharmacologic activity.

Conjugates of catecholamines. 5. Synthesis and beta-adrenergic activity of N-(aminoalkyl)norepinephrine derivatives.

A novel series of N-aminoalkyl congeners and model derivatives of norepinephrine has been synthesized. Compounds that were structurally related to epinephrine were prepared from fully protected intermediates. Alternatively, isoproterenol-related compounds were synthesized via reductive amination of preformed methyl ketone derivatives with norepinephrine. The beta-adrenergic activities of these new compounds were assessed through measurement of intracellular cyclic AMP accumulation in S49 mouse lymphoma cells and displacement of iodocyanopindolol (ICYP) from membrane preparations. Congeners that contained an underivatized primary amine function exhibited virtually no activity in these assays. However, when this amine function was acylated (e.g., to an amide, carbamate, urea, sulfonamide, etc.), the products exhibited generally increased beta-adrenergic activity, which was, however, strongly dependent on the nature of the acylating group and also the length of the spacer. In particular, a benzyl carbamate derivative containing a branched, seven-carbon spacer group was 40 times more potent than isoproterenol in the in vitro S49 assay.

Pharmacologic effects of autacoids on subsets of T cells. Regulation of expression/function of histamine-2 receptors by a subset of suppressor cells.

Autacoids (principally histamine, beta adrenergic catecholamines, and prostaglandins E and A) have only recently been recognized as substantive moderators of a number of immune functions. If autacoids are to be considered as potential therapeutic immunomodulators, it is necessary to understand their effects on subsets of T cells while they are and are not in contact with each other. This report demonstrates that autacoid receptors are nonrandomly distributed on phenotypically and functionally distinct subsets of human T cells. Each human T cell subset responded to both histamine and isoproterenol, but the dose response curve and maximal efficacy varied widely between the subsets. The suppressor T cells were more responsive to both histamine and isoproterenol than helper/inducer T cells (TH) or cytotoxic T cells (Tc). We found that after mitogenic stimulation the response to histamine, but not isoproterenol, was greatly increased only in TH (Leu 3+) and Tc (Leu 2+, 9.3+) subsets, and that this effect may be regulated by suppressor T cells (Leu 2+, 9.3-). The dramatic rise in cAMP accumulation in response to histamine in mitogen-treated TH and Tc was totally blocked by an H2 antagonist (cimetidine), but not by an H1 antagonist (mepyramine). These findings indicate interdependence of (a) immunologically uncommitted subsets in their response to selected drugs, and (b) control of basal- and autacoid-induced cAMP production, as well as (c) increased qualitative and quantitative selectivity, which is caused by mitogen. If we had performed these experiments only on unseparated cells we would not have observed the remarkable selectivity of autacoid effects on subsets of T cells.

Conjugates of catecholamines. II. In vitro and in vivo pharmacological activity of N-alkyl-functionalized carboxylic acid congeners and amides related to isoproterenol.

In this study, 10 congeners of isoproterenol were systematically synthesized and pharmacologically tested in both in vitro and in vivo systems. The aim was to produce compounds that were more potent and had effects different from those of the parent compound and that could ultimately be attached covalently to inert peptide carriers offering versatility in size, pK, and other physicochemical properties. The congeners synthesized were tested in the S49 mouse lymphoma assay for their ability to stimulate cyclic AMP accumulation and were shown to have potencies relative to isoproterenol ranging from 4 orders of magnitude less potent to 4 orders of magnitude more potent than isoproterenol in eliciting this response. Some of the congeners were also tested in a guinea pig isolated atrial preparation, a rat blood pressure assay, a guinea pig bronchodilation assay, and an anesthetized dog preparation. In these assays, the congeners were shown to have the same types of activities as in the S49 cell assay. The results of these studies indicate that structural modifications distant from the catecholamine moiety dramatically alter the pharmacological profile of the congeners versus the parent compound, resulting in a series of ligands with a wide range of activities.

Conjugates of catecholamines. IV. In vitro and in vivo pharmacological activity of monodisperse oligopeptide conjugates.

Conjugates of low molecular weight amines with inert peptide carriers can be made to preserve some pharmacologic effects of the ligand. No previous studies of conjugates have systematically derivatized the ligand to optimize its activity or affinity to receptors; or have pharmacophores been selected for conjugation on the basis of their effects making them particularly interesting as ligands. Chemically pure and characterizable conjugates ranging from single blocked amino acid derivatives to isomeric pentapeptides were constructed and tested for beta adrenergic properties. Sixteen conjugates with varying water solubility, amino acid composition and sequence, charged groups and spacer length between ligand and carrier showed potencies and duration of action widely different from isoproterenol. In the in vitro S-49 mouse lymphoma assay a range of relative potencies (as compared to isoproterenol) from 10(-6) to 1 time as potent was obtained, whereas in the in vivo rat blood pressure assay these compounds were shown to be from one-fifth to 4 times as potent as isoproterenol. The most potent compound tested, Boc-Phe(NH)-Gly-NHCH3, was also tested in a guinea-pig atrial preparation as well as in an anesthetized cardiovascular dog preparation. The results indicated that the compound was approximately 3.5 times more potent in producing an inotropic response than isoproterenol. In some instances, ostensively trivial changes quite distant from the pharmacophores in amino acid sequence of the carrier made major changes in potency and efficacy of the compound.

Conjugates of catecholamines. 1. N-alkyl-functionalized carboxylic acid congeners and amides related to isoproterenol.

A series of functionalized catecholamines (congeners) has been synthesized in which, formalistically, the N-isopropyl group of isoproterenol has been extended by a linear alkyl chain of varying length, terminated by a carboxy group or a substituted amide. The compounds were prepared generally via the reductive amination of norepinephrine with a keto acid or a preformed keto amide. An alternate synthesis of the model amide derivatives, involving activation of the carboxylic acid congeners and coupling with amines, was complicated in the case of short-chain derivatives by facile cyclization to lactams. In vitro evaluation of these compounds as potential beta-adrenergic agonists has shown that, while the carboxylic acid congeners have relatively low potencies, the model amide derivatives have potencies that are highly dependent on both the length of the alkyl chain and also the nature of the substituent on the amide. In general, aromatic amides are the most potent, although the nature and position of substituents on the aromatic group dramatically influences their potency. The implications of these studies, in terms of general beta-adrenergic drug design and also the attachment of the carboxylic acid congeners to carriers, are discussed.

Autacoids as modulators of the inflammatory and immune response.

Once considered only mediators of inflammation, autacoids, (histamine, prostaglandins and beta-mimetic catecholamines) have been found to be generated during specific early and late phases of immunity. They need sufficient concentrations to affect immunocytes and can modulate immunity usually by inhibiting it. Receptors for the autacoids on the immunocytes are nonrandomly distributed. A small portion of T suppressor cells always appear to have receptors on them, but precursor B cells and precursors of T cells that produce lymphokines or are responsible for cytolysis do not. Instead, as these cells mature they develop their autacoid receptors. With one exception, the function of the immunocytes is inhibited by the effects of autacoids. Again, in all but one instance, that inhibitory modulating effect is mediated by and directly proportional to the intracellular concentrations of cyclic adenosine monophosphate (AMP) generated by the autacoid. The clinical implications of these observations are beginning to be appreciated. One of them is that pharmacologic antagonists of the autacoids can have predictable but hitherto unanticipated effects on immune functions. It is inconceivable that these effects will not have clinical value.