Localization of histamine and histamine H2-receptor antagonists in the gastric mucosa.

Synopsis Histamine stimulates acid secretion by the parietal cell and this secretion is inhibited by the histamine H₂-receptor antagonists. Whole body autoradiography showed that radioactivity from¹⁴C-histamine was localized in the artery walls of the stomach and in the muscularis mucosae, but that the level in the fundic mucosa was the same as the blood.When the H₂-receptor antagonists burimamide, metiamide and cimetidine were labelled with³⁵S,¹⁴C or³H and dosed to rats, whole body autoradiography showed that the stomach was predominantly labelled in the glandular mucosa from 5 to 120 min after administration. Microautoradiography in the rat and dog after intravenous injection of [³H] metiamide or [³H] cimetidine demonstrated an uptake of tritium in the parietal cell cytoplasm that was 3- to 4-times greater than that found in adjacent peptic cells or areas of muscularis mucosa. The preferential labelling persisted at a low level up to 6 h after injection in the rat. The localization of radioactivity from the H₂-antagonists in the parietal cell cytoplasm correlates well with their pharmacological activity in preventing acid secretion from this cell.     

Endothelin causes contraction of human esophageal muscularis mucosae through interaction with both ETA and ETB receptors

Endothelin (ET) causes contraction of the muscularis mucosae in the guinea pig esophagus, but its role in the human esophagus remains unknown. To investigate effects of ET in the human esophagus, we measured contraction of isolated human esophageal muscularis mucosae strips caused by ET related peptides and binding of 125I-ET-1 to cell membranes prepared from the human esophageal muscularis mucosae. Autoradiography demonstrated specific binding of 125I-ET-1 to the muscularis mucosae and muscularis propria (muscularis externa) of the human esophagus. ET-1 caused tetrodotoxin and atropine-insensitive contraction of muscularis mucosae strips. In terms of the maximal tension of contraction, ET-1 and ET-2 were equal in efficacy. The relative potencies for ET related peptides to cause contraction were ET-1=ET-2>ET-3>sarafotoxin S6c (SX6c), an ETB receptor agonist. ET-1 caused contraction was mildly inhibited by BQ-123, an ETA receptor antagonist, and not by BQ-788, an ETB receptor antagonist. It was moderately inhibited by the combination of both antagonists, indicating synergistic inhibition. Furthermore, desensitization to SX6c with SX6c pretreatment failed to abolish the contractile response to ET-1, which was completely inhibited by BQ-123. These indicate the involvement of both ETA and ETB receptors in the contraction. Binding of 125I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ETA and ETB receptors. This study demonstrates that, the muscularis mucosae of the human esophagus, similar to that of the guinea pig esophagus, possesses both ETA and ETB receptors mediating muscle contraction.     

Potential anti-diabetic applications of a new molecule with affinity for β3-Adrenoceptors

β₃-adrenoceptors have been described through both molecular and pharmacological studies. In this context, it has been characterized the β₃-adrenoceptors agonist activity of a new compound (Trecadrine^®), and evaluated whether it exhibits additional activity at β₁ and β₂- adrenoceptor subtypes. In addition, it has been tested its potential anti-diabetic properties in a model of alloxan-induced diabetes in rats. Our data show that Trecadrine^® induces oesophageal muscularis mucosae relaxation, indicating a putative action on β₃-adrenoceptors. In the absence of β₃-adrenoceptors antagonists, assays with β₁ and β₂-adrenergic antagonists reveal a quite remarkable selectivity for β₃-adrenoceptors. Furthermore, it has been suggested that this molecule has hypoglycaemic and lipomobilizing effects, although hypoglycaemia was not related to an increase in insulin secretion in diabetic rats. It is concluded that Trecadrine^® mainly acts through β₃-adrenoceptors, and it may constitute a potential drug in the treatment of diabetes.     

Design,synthesis and biological evaluation of novel hydrogen sulfide releasing capsaicin derivatives

Capsaicin (CAP), the prototypical TRPV1 agonist, is the major active component in chili peppers with health-promoting benefits. However, its use is limited by the low bioavailability and irritating quality. In this study, for improving the activity of CAP and alleviating its irritating effects, a series of H₂S-releasing CAPs were designed and synthesized by combining capsaicin and dihydro capsaicin with various hydrogen sulfide donors. The resulting compounds were evaluated their TRPV1 agonist activity, analgesic activity, anticancer activities, H₂S-releasing ability, and gastric mucosa irritation. Biological evaluation indicated that the most active compound B₉, containing 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione moiety as H₂S donor, had better analgesic activity and displayed more potent cytotoxic effects on the test cell lines than the lead compound CAP. Furthermore, the preferred compound, B₉ reduced rat gastric mucosa irritation caused by CAP. Notably, the improved properties of this derivative are associated with its H₂S-releasing capability.     

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.     

Specific activation of cyclic AMP-dependent protein kinase(s) by H2-histamine agonists in isolated gastric mucosal cells from guinea-pig

Histamine stimulated cyclic AMP-dependent protein kinase activity in dispersed mucosal cells from guinea-pig gastric fundus (K_a = 5 μM). The H₂-agonists dimaprit and impromidine produced similar effects, while the H₁-agonist 2-(2-pyridyl) ethylamine had only a weak one. The H₂-antagonist cimetidine competitively inhibited 0.1 mM histamine stimulation (K_i = 2 μM). In contrast, the H₁-antagonist diphenhydramine had no effect up to 1 mM.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

Two classes of endothelin receptors mediating contraction in esophageal muscularis mucosae

Endothelin (ET) causes contraction of the esophageal muscularis mucosae. To characterize the ET receptor subtypes involved in contraction, we measured contraction of isolated muscularis mucosae strips caused by ET-related peptides and binding of (125)I-ET-1 to cell membranes prepared from the guinea pig esophageal muscularis mucosae. Autoradiography demonstrated (125)I-ET-1 binding to the muscularis mucosae and muscularis propria. ET-1 caused tetrodotoxin and atropine-insensitive contraction of esophageal muscularis mucosae strips. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. FR-139317, an ET(A) receptor antagonist, or BQ-788, an ET(B) receptor antagonist, alone did not alter responses to ET-1. However, the combination of both antagonists almost abolished the ET-1-induced contraction, indicating synergistic inhibition. Desensitization to sarafotoxin S6c, an ET(B) receptor agonist, failed to abolish the response to ET-1, which was completely inhibited by FR-139317. These indicate the involvement of both ET(A) and ET(B) receptors in the contraction. Binding of (125)I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ET(A) and ET(B) receptors.This study demonstrates that the esophageal muscularis mucosae possesses both ET(A) and ET(B) receptors mediating muscle contraction. There is cooperation between these two subtypes of ET receptors in the esophagus mediating muscle contraction.     

Role of histamine in the antitumour activity of endotoxin

Summary The role of histamine in the antitumour activity of endotoxin against solid syngeneic Meth-A sarcoma in BALB/c mice was studied. Endotoxin induces haemorrhagic necrosis and regression of this tumour. Histamine and the selective H₁ receptor agonist 2-pyridylethylamine mimicked the induction of necrosis but did not cause regression. The selective H₂ receptor agonist dimaprit did not cause any tumour damage. The effect of histamine could be inhibited by the H₁ receptor antagonists diphenhydramine and promethazine but not by the H₂ receptor antagonist cimetidine. Endotoxin-induced necrosis was slightly affected by diphenhydramine, and the incidence of regression was reduced by both H₁ antagonists. Cimetidine potentiated endotoxin-induced regression. Similar effects were observed concerning the effects of H-receptor antagonists on necrosis and regression induced by tumour necrosis serum (TNS). Histological examination revealed no marked additional effects of diphenhydramine or cimetidine on endotoxin-induced hyperaemia, haemorrhagic necrosis, and mitotic arrest of the tumour cells. Only cimetidine increased the extent of nonhaemorrhagic necrosis. The endotoxin-induced release of tumour necrosis factor and cytostatic activity in TNS was clearly reduced by diphenhydramine, but hardly affected by cimetidine.Data indicate that intact H₁ receptors are required for the induction of tumour regression and antitumour factors by endotoxin. Concomitant H₂ blockade may facilitate this by stimulating H₁ receptor-mediated processes upon endotoxin-induced histamine release, although a cimetidine-induced inhibition of T-suppressor cell activation might also be involved.     

Synthesis and characterization of a peripherally restricted CB1 cannabinoid antagonist,URB447, that reduces feeding and body-weight gain in mice

Cannabinoid CB₁ receptor antagonists reduce body weight in rodents and humans, but their clinical utility as anti-obesity agents is limited by centrally mediated side effects. Here, we describe the first mixed CB₁ antagonist/CB₂ agonist, URB447 ([4-amino-1-(4-chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl](phenyl)methanone), which lowers food intake and body-weight gain in mice without entering the brain or antagonizing central CB₁-dependent responses. URB447 may provide a useful pharmacological tool for investigating the cannabinoid system, and might serve as a starting point for developing clinically viable CB₁ antagonists devoid of central side effects.     

Evidence that ICI 118, 551 is a potent, highly Beta 2-selective adrenoceptor antagonist and can be used to characterize Beta-adrenoceptor populations in tissues

pA₂ values for a new ß-adrenoceptor antagonist, ICI 118, 551 (erythro-DL-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-o1), have been obtained on intrinsic tone tracheal chain preparations and on spontaneously beating atrial preparations from guinea-pigs. Two different agonists, fenoterol (ß₂-selective) and noradrenaline (ß₁-selective) were used. The slopes of the Schild plots were not significantly different from 1.0. The antagonist was very potent (pA₂ on trachea, fenoterol as agonist, was 8.69) and also highly ß₂-selective (pA₂ on atria, noradrenaline as agonist, was only 6.96). On guinea-pig trachea (which contains ß₁- and ß₂-adrenoceptors) the potency was 44 times higher when fenoterol was the agonist than when noradrenaline was the agonist. On guinea-pig atria (which contains only ß₁-adrenoceptors) the pA₂ value was the same whichever agonist was used. Thus ICI 118, 551 has been shown to be potent and the most ß₂-selective antagonist so far studied in our laboratory. In experiments carried out with selective agonists ICI 118, 551 distinguished clearly between tissues with a mixed ß-adrenoceptor population (different pA₂ values) and those with a homogeneous population (single pA₂ value). Therefore, ICI 118, 551 is a valuable addition to the group of ß₂-selective adrenoceptor antagonists.     

Synthesis and evaluation of arylpiperazines derivatives of 3,5-dioxo-(2H,4H)-1,2,4-triazine as 5-HT1AR ligands

5-HT_1AR agonist or partial agonists are established drug candidates for psychiatric and neurological disorders. We have reported the synthesis and evaluation of a series of high affinity 5-HT_1AR partial agonist PET imaging agents with greater selectivity over α-1AR. The characteristic of these molecules are 3,5-dioxo-(2H,4H)-1,2,4-triazine skeleton tethered to an arylpiperazine unit through an alkyl side chain. The most potent 5-HT_1AR agonistic properties were found to be associated with the molecules bearing C-4 alkyl group as the linker. Therefore development of 3,5-dioxo-(2H,4H)-1,2,4-triazine bearing arylpiperazine derivatives may provide high affinity selective 5-HT_1AR ligands. Herein we describe the synthesis and evaluation of the binding properties of a series of arylpiperazine analogues of 3,5-dioxo-(2H,4H)-1,2,4-triazine.     

Synthesis and pharmacological investigation of azaphthalazinone human histamine H1 receptor antagonists

5-Aza, 6-aza, 7-aza and 8-aza-phthalazinone, and 5,8-diazaphthalazinone templates were synthesised by stereoselective routes starting from the appropriate pyridine/pyrazine dicarboxylic acids by activation with CDI, reaction with 4-chlorophenyl acetate ester enolate to give a β?ketoester, which was hydrolysed, and decarboxylated. The resulting ketone was condensed with hydrazine to form the azaphthalazinone core. The azaphthalazinone cores were alkylated with N-Boc-D-prolinol at N-2 by Mitsunobu reaction, de-protected, and then alkylated at the pyrrolidine nitrogen to provide the target H₁ receptor antagonists. All four mono-azaphthalazinone series had higher affinity (pK_i) for the human H₁ receptor than azelastine, but were not as potent as the parent non-aza phthalazinone. The 5,8-diazaphthalazinone was equipotent with azelastine. The least potent series were the 7-azaphthalazinones, whereas the 5-azaphthalazinones were the most lipophilic. The more hydrophilic series were the 8-aza series. Replacement of the N-methyl substituent on the pyrrolidine with the n-butyl group caused an increase in potency (pA₂) and a corresponding increase in lipophilicity. Introduction of a β-ether oxygen in the n-butyl analogues (2-methoxyethyl group) decreased the H₁ pA₂ slightly, and increased the selectivity against hERG. The duration of action in vitro was longer in the 6-azaphthalazinone series. The more potent and selective 6-azaphthalazinone core was used to append an H₃ receptor antagonist fragment, and to convert the series into the long acting single-ligand, dual H₁ H₃ receptor antagonist 44. The pharmacological profile of 44 was very similar to our intranasal clinical candidate 1.     

Histamine H2-receptors in the gastric mucosa: role in acid secretion.

Currently, two major hypotheses dominate thinking about the role of histamine in the regulation of gastric acid secretion. Code has proposed that histamine is the final common mediator of secretagogue action on the parietal cell while Konturek and Grossman have suggested a multi-receptor control of the secretory process. Experimental results derived from the use of recently synthesized histamine H₂-receptor antagonists have been used by both groups to support their hypotheses. Paradoxically, these hypotheses depend on the presumed specificity of the H₂-antagonists in blocking histamine mediated acid secretion while the apparent lack of such secretagogue specificity of the H₂-antagonists is an important basis for the development of the hypotheses. Our review will analyze the experimental evidence which implicates the histamine H₂-receptor in the control of hydrogen ion secretion as well as evidence for and against receptor specificity in the gastric mucosa of histamine H₂-receptor antagonists.     

Synthesis and analgesic effects of novel β2-tryptophan hexapeptide analogs

Aiming to develop more potent analgesic substances a new series of hexapeptides containing β²-tryptophan analogues was synthesized. The Trp in position 4 and 5, respectively in Ac-Arg-Phe-Met-Trp-Met-Lys-NH₂ (opioid receptor antagonist) and Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ (highly potent and selective NOP-receptor agonist) was substituted by the (S)-2-(1-methyl-1H-indol-3-yl)propionic residue or the (S)-2-(5-methoxy-1H-indol-3-yl)propionic residue. The analgesic effect of the four newly synthesized compounds has been evaluated in male Wistar rats by PP- and HP tests and compared to the native templates. Further estimation of the mechanisms of action of each compound was achieved using specific antagonists—naloxone for opioid and JTC801 for the NOP receptor. Replacement of Trp with β²-tryptophan analogues in 4th position (Ac-Arg-Phe-Met-Trp-Met-Lys-NH₂) led to increased and longer lasting analgesic effect. The results obtained permit us to assume that both opioid and NOP receptors take part in the newly synthesized compounds analgesic effects.     

Highly selective vagotomy and duodenal ulcers that fail to respond to H2 receptor antagonists

A study was conducted to see whether patients with duodenal ulcers that failed to heal in response to H₂ receptor antagonists had a higher incidence of recurrent ulceration after highly selective vagotomy than patients whose ulcers healed with these drugs. Between 1977 and 1983, 157 patients had a highly selective vagotomy for uncomplicated duodenal ulcer; in 57 patients the ulcer had failed to heal despite treatment with H₂ receptor antagonists (refractory group), 19 patients had developed recurrent ulceration while receiving maintenance treatment, 67 patients had remained healed while taking H₂ receptor antagonists but suffered frequent relapses when treatment was stopped, and 14 patients had not been given these drugs before operation. The overall incidence of recurrent ulceration was 6% after two years and 11% after five years of follow up. In the refractory group, however, the incidence of recurrent ulceration was 18% at two years and 34% after five years, whereas the incidence of recurrence was only 1.5% at two years and 3% after five years in patients whose ulcers had healed with H₂ receptor antagonists. Resistance to H₂ receptor antagonists was not related to preoperative basal or peak acid output but was related to cigarette smoking. Factors associated with recurrent ulceration after highly selective vagotomy were basal acid outputs before and after operation, cigarette smoking, and the surgeon who performed the operation.Duodenal ulcers that fail to respond to H₂ receptor antagonists represent a more severe ulcer diathesis, for which highly selective vagotomy is less effective.     

2,4-Diaminopyrimidines as histamine H4 receptor ligands—Scaffold optimization and pharmacological characterization

The human histamine H₄ receptor (hH₄R) is a promising new target in the therapy of inflammatory diseases and disorders of the immune system. For the development of new H₄R antagonists a broad ligand-based virtual screening was performed resulting in two hits. The dissection of their common annelated aromatic core into its heteromonocyclic components showed that 2,4-diaminopyrimidine is a potent hH₄R affinity scaffold, which was comprehensively investigated. Structure–activity relationship studies revealed that slight structural changes evoke extensive differences in functional activities and potencies: while o- and p-substituted benzyl amines mainly showed partial agonism, m-substituted and rigidified ones exhibited inverse agonist efficacy.     

The effect of pKa on pyrimidine/pyridine-derived histamine H4 ligands

During the course of our efforts toward the discovery of human histamine H₄ antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H₄ receptor. This observation was further investigated by synthesizing a series of ligands that varied in pK_a of the pyrimidine derived H₄ ligands by over five orders of magnitude and the effect on histamine H₄ affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H₄ receptor antagonists.     

Sensitization by Pulmonary Reactive Oxygen Species of Rat Vagal Lung C-Fibers: The Roles of the TRPV1, TRPA1, and P2X Receptors

Sensitization of vagal lung C-fibers (VLCFs) induced by mediators contributes to the pathogenesis of airway hypersensitivity, which is characterized by exaggerated sensory and reflex responses to stimulants. Reactive oxygen species (ROS) are mediators produced during airway inflammation. However, the role of ROS in VLCF-mediated airway hypersensitivity has remained elusive. Here, we report that inhalation of aerosolized 0.05% H₂O₂ for 90 s potentiated apneic responses to intravenous capsaicin (a TRPV1 receptor agonist), α,β-methylene-ATP (a P2X receptor agonist), and phenylbiguanide (a 5-HT₃ receptor agonist) in anesthetized rats. The apneic responses to these three stimulants were abolished by vagatomy or by perivagal capsaicin treatment, a procedure that blocks the neural conduction of VLCFs. The potentiating effect of H₂O₂ on the apneic responses to these VLCF stimulants was prevented by catalase (an enzyme that degrades H₂O₂) and by dimethylthiourea (a hydroxyl radical scavenger). The potentiating effect of H₂O₂ on the apneic responses to capsaicin was attenuated by HC-030031 (a TRPA1 receptor antagonist) and by iso-pyridoxalphosphate-6-azophenyl-2′,5′-disulphonate (a P2X receptor antagonist). The potentiating effect of H₂O₂ on the apneic responses to α,β-methylene-ATP was reduced by capsazepine (a TRPV1 receptor antagonist), and by HC-030031. The potentiating effect of H₂O₂ on the apneic responses to phenylbiguanide was totally abolished when all three antagonists were combined. Consistently, our electrophysiological studies revealed that airway delivery of aerosolized 0.05% H₂O₂ for 90 s potentiated the VLCF responses to intravenous capsaicin, α,β-methylene-ATP, and phenylbiguanide. The potentiating effect of H₂O₂ on the VLCF responses to phenylbiguanide was totally prevented when all antagonists were combined. Inhalation of 0.05% H₂O₂ indeed increased the level of ROS in the lungs. These results suggest that 1) increased lung ROS sensitizes VLCFs, which leads to exaggerated reflex responses in rats and 2) the TRPV1, TRPA1, and P2X receptors are all involved in the development of this airway hypersensitivity.     

Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

The H₂-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, ²H¹H exchange from a mixture of ²H₂ and ¹H₂ in presence of ²H₂O and ¹H₂O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H₂ oxidation reaction, there was no significant discrimination between ²H₂ and ¹H₂, indicating that the initial H₂-activation step in the over-all H₂ oxidation reaction is not rate-limiting. By use of improved methods, an apparent K_m for H₂ of 0.05 micromolar was determined. The H₂ oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H₂ oxidation and stimulated O₂ uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H₂ to O₂. Partial pressures of H₂ at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO₂ evolution by low partial pressures of H₂ suggests that H₂ utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H₂ uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO₂ (initial concentration) in solution inhibited H₂ uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H₂ uptake by succinate, acetate, formate, and CO₂ in the metabolism of the H₂-uptake-positive strains of Rhizobium.