Inosine Triphosphate Research Articles

Lack of association between the ITPA 94C>A polymorphism and adverse effects from azathioprine

A 94C>A missense mutation in the ITPA gene which encodes inosine triphosphate pyrophosphatase has been associated with adverse effects from azathioprine, specifically flu-like symptoms, pancreatitis and rash. We hypothesized that this association may also be present in a larger, population-based group of inflammatory bowel disease patients intolerant of thiopurine drugs. We performed genotyping for this polymorphism and TPMT*2 and TPMT*3 in 73 such patients and 74 patients with inflammatory bowel disease who have tolerated azathioprine. We could not demonstrate a significant association between the ITPA94C>A genotype and any adverse effects (Odds ratio (OR) 1.015, 95% confidence interval (CI) 0.360-2.867, P = 0.593), flu-like symptoms (OR 1.547, 95%CI 0.368-6.496, P = 0.398), rash (no ITPA 94C>A polymorphism identified) or pancreatitis (no ITPA 94C>A polymorphism identified). We found no significant association between the ITPA 94C>A polymorphism and adverse effects to thiopurine drugs.

Distribution of ITPA P32T alleles in multiple world populations.

Dose-limiting toxicity from azathioprine treatment affects up to 37% of patients. Screening for thiopurine methyltransferase (TPMT) polymorphisms will prospectively identify approximately 10% of patients. Recently, a polymorphism in the inosine triphosphate pyrophosphatase gene (ITPA) has been associated with severe azathioprine toxicity. We demonstrate here that this proline to threonine substitution at codon 32 in the ITPA gene is found at low frequency in Central/South American populations (1-2%), at a constant frequency across Caucasian and African populations (6-7%), and is highest in Asian populations (14-19%). This data is consistent with previously described allele frequencies in other Caucasian (7%), African (5%), and Asian (11-15%) populations. This data provides a foundation on which prospective screening studies can be planned to identify patients at risk for severe toxicity from azathioprine therapy.

Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes.

Earlier it has been shown that human proliferating/undifferentiated basal keratinocytes hold the full capacity for autocrine catecholamine synthesis/degradation and express beta2-adrenoceptors (beta2-AR). In this report, we show that human melanocytes also express all of the mRNA and enzymes for autocrine synthesis of norepinephrine but fail to produce epinephrine. So far, it was established that human melanocytes express alpha1-AR which are induced by norepinephrine yielding the inosine triphosphate diacylglycerol signal. The presence of catecholamine synthesis and the beta2-AR signal escaped definition at that time. Using RT-PCR, immunofluorescence and radioligand binding with the beta2-AR antagonist (-)-[3H]CGP 12177, we show here that human melanocytes express functional beta2-AR (4230 receptors per cell) with a Bmax at 129.3 and a KD of 3.19 nM but lack beta1-AR expression. beta2-AR stimulation with epinephrine 10(-6) M and salbutamol 10(-6)-10(-5) M yielded a strong cyclic adenosine monophospate (cAMP) response in association with upregulated melanin production. Taken together these results indicate that the biosynthesis and release of epinephrine (10(-6) M) by surrounding keratinocytes can provide the cAMP response leading to melanogenesis in melanocytes via the beta2-AR signal. Moreover, the discovery of this catecholaminergic cAMP response in melanocytes adds a new source for this important second messenger in melanogenesis.

Identification of two novel tumor-associated antigens recognized by HLA-B46-restricted cytotoxic T lymphocytes

The objective of this study was to identify novel genes and peptides capable of inducing tumor-reactive cytotoxic T lymphocytes (CTLs) in cancer patients with an HLA-B46 allele, which is preferentially expressed in Asians. We show that two genes encoding splicing factor (SF) 2 and inosine triphosphate pyrophosphatase (ITPA) have epitopes recognized by HLA-B46-restricted and tumor cell-reactive CTL lines established from tumor-infiltrating lymphocytes of colon cancer. The SF2 is essential for constitutive pre-mRNA splicing, while the enzyme ITPA controls nucleotide levels. The mRNA expression levels of these genes were higher in tumor cells than those in normal tissues. Five peptides, three from SF2 and two from ITPA, had the ability to induce HLA-B46-restricted and peptide-specific CTLs reactive to tumor cells in peripheral blood mononuclear cells of cancer patients. These results may provide new information for better understanding of host-tumor interaction at the molecular level and the development of peptide-based immunotherapy for HLA-B46+ cancer patients.

Backtracking by single RNA polymerase molecules observed at near-base-pair resolution.

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

DNA polymorphisms in ITPA including basis of inosine triphosphatase deficiency.

Intracellular concentrations of the nucleotide inosine triphosphate (ITP) are regulated by ITP-ase (EC 3.6.1.19), which is encoded by ITPA on chromosome 20p. Subjects with complete deficiency of ITP-ase activity (MIM 147520) have elevated ITP concentrations in erythrocytes, but no obvious clinical abnormalities. Based on biochemical screening, complete ITP-ase deficiency has been postulated to result from homozygosity for a dysfunctional allele, with an estimated frequency of 0.05 in Caucasians. ITP-ase deficiency has not yet been characterized at the molecular genetic level. Sequencing of the genomic DNA from a Caucasian subject with complete ITP-ase deficiency revealed homozygosity for missense mutation 198C>A, which predicted a threonine for proline substitution at codon 32 (P32T), whereas among 125 normal Caucasians, there were no homozygotes for P32T (P = 0.0079). The P32T allele frequency of 0.07 in Caucasians was similar to the estimates derived from earlier biochemical studies. P32T was found to be present at varying frequency in other ethnic groups. Two common synonymous single-nucleotide polymorphisms were also identified. These ITPAmarkers, including P32T, provide tools for further study of association with clinical and biochemical phenotypes.

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G-->A, 561G-->A, 708G-->A) and two associated with ITPase deficiency (94C-->A, IVS2+21A-->C). Homozygotes for the 94C-->A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C-->A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A-->C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C-->A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A-->C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A-->C heterozygotes and 94C-->A/IVS2+21A-->C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

ChemInform Abstract: Acid—Base Properties of the 5′‐Triphosphates of Guanosine and Inosine (GTP4‐ and ITP4‐) and of Several Related Nucleobase Derivatives.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPAGene

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

As to the clastogenic-, sister-chromatid exchange inducing-and cytotoxic activity of inosine triphosphate in cultures of human peripheral lymphocytes

The influence of commercial inosine triphosphate (ITP) on the chromosome aberration rate, the mitotic rate, sister-chromatid exchange (SCE) frequency, and the proportion of first (X1), second (X2) and third (X3) division metaphases was investigated in 72 h cultures of human peripheral lymphocytes. The blood donors had mild inactive arthrosis and a normal health check-up. All cultures of each volunteer were set-up simultaneously. In contrast to a previous report [Arch. Biochem. Biophys. 278 (1990) 238–244], it was demonstrated in two preliminary studies (number of subjects, n=5 each) that ITP at a final concentration of 100 μM does not induce chromosomal aberrations and, furthermore, that not ITP concentrations higher than 100 μM but ITP doses higher than 3.8 mM prohibit culture growth. Based on these results, cultures with a final ITP concentration of 3.6 mM (max.) and 1.8 mM (max./2) were compared with control cultures (number of subjects n=10; three males and seven females, mean age x=57.6 years). Whereas no increase in the chromosomal breakage rate was observed in cultures with an ITP concentration of 1.8 mM and only a marginally significant one ( P=0.048) for 3.6 mM ITP cultures, a highly significant induction of SCEs, not only at an ITP concentration of 3.6 mM ( P<0.0001) but also at 1.8 mM ( P<0.0001) was seen. The increase in the SCE frequency was not linear, but steeper from 0 to 1.8 mM than from 1.8 to 3.6 mM. Nevertheless, the difference between 1.8 and 3.6 mM cultures was significant ( P=0.027). The distribution of the number of SCEs per metaphase as well as the distribution of SCEs per chromosome correspond to the expected Poisson values. The investigation of the cytotoxic effect of the studied ITP concentrations revealed a highly significant reduction of the mitotic rate from 0 to 1.8 mM as well as from 1.8 to 3.6 mM in the aberration studies (all P values are equal to smallest possible one for a sample size of 10, namely, 0.002), and in the SCE studies there is a significant decrease in the X3 frequency when ITP is increased (0–1.8 mM: P=0.0061 and 1.8–3.6 mM: P<0.0001). The proportion of X1 within all X1 and X2 metaphases changes significantly only at the second dose step (0–1.8 mM ITP: P=0.22 and 1.8–3.6 mM ITP: P<0.0001). The results are discussed.

Acid–base properties of the 5′-triphosphates of guanosine and inosine (GTP4− and ITP4−) and of several related nucleobase derivatives

The acidity constants of H2(NTP)2− [NTP4− = guanosine 5′-triphosphate (GTP4−) or inosine 5′-triphosphate (ITP4−)], which may lose three protons giving (NTP − H)5−, were determined by potentiometric pH titrations (25 °C; I = 0.1 M, NaNO3) and values for the deprotonation of H3(NTP)− were estimated via micro-acidity-constant evaluations. Complete micro-acidity-constant schemes for both H3(GTP)− and H3(ITP)− are given. From the evaluations it follows that the ratios, R, of the twofold protonated and isocharged species (H·NTP·H)2− and (NTP·H2)2− for ITP and GTP, which carry one proton at N7 and one at the terminal γ-phosphate group or both protons at the triphosphate chain, respectively, are about 1 ∶ 1 for the ITP system and about 10 ∶ 1 for the GTP system. This confirms the higher basicity of N7 in the guanosine residue compared with that in the inosine moiety. For the evaluation of the indicated analysis it was also necessary to consider the acid–base properties of guanosine, inosine, adenosine, and of the 5′-triphosphates of adenosine, cytidine, uridine and thymidine; the corresponding acidity constants were in part taken from our earlier work and in part are also measured now.

Inhibition of rat parotid ecto-ATPase activity

The inhibitory profile of several known and suspected ecto-ATPase inhibitors was compared on ecto-ATPase activity in rat parotid plasma membranes. Those chemicals with high IC 50 (above 130 μM) were the nucleotides α,β-methylene ATP, β,γ-methylene ATP, 2-methylthio ATP, inosine triphosphate, 5′- p-fluorosulphonylbenzoyladenosine, the sulphonates, 1-amino-2-naphthol-4-sulphonic acid, Coomassie brilliant blue G, and the stilbene disulphonates, DIDS and SITS. Those agents with low IC 50 were: Coomassie brilliant blue R (114 μM), ATPγS (49 μM), suramin (72 μM) and Reactive blue 2 (28 μM). The last three inhibitors have similar potencies as inhibitors of ATP hydrolysis by whole parotid acinar cells. ARL67156, a selective inhibitor of ecto-ATPase, had an IC 50 of approx. 120 μM. Suramin displayed non-competitive inhibition of ecto-ATPase whereas the inhibitory effects of ATPγS and Reactive blue 2 were curvilinear on Dixon plots. These results define the effects of various agents on ecto-ATPase in an exocrine tissue that has been shown to respond to extracellular ATP.

Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate.

To identify stable RNA secondary structure causing band compression, 30 lambda DNA clones and four cDNA clones (about 10 kb in total length) were sequenced using Transcriptional Sequencing, which is based on the phage RNA polymerase chain termination reaction with fluorescent 3' deoxynucleoside triphosphate, using the canonical set of rNTPs for the substrate. Electrophoresis was performed on acrylamide gel containing 7 M urea at 50 degrees C using ABI 377 DNA sequencer. A total of 159 band compressions were identified, and most compression sites seem to be due to hairpin structures. We also found that the presence of rITP in place of rGTP in the sequencing reaction can entirely eliminate all band compressions. The use of rITP gave a better peak uniformity and resolution in the sequencing gel in the case of lambda DNA than with c7rGTP, leading to improved accuracy in the sequence determination. Substitution of the base analog rITP for rGTP should be useful for accurate sequencing determination.

Role of Guanosine Triphosphate in Ferric Ion-Linked Fenton Chemistry

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Force development with inosine triphosphate and uridine triphosphate in chemically skinned vascular smooth muscle

The contraction of vascular smooth muscle is thought to be regulated by reversible phosphorylation of the 20,000 dalton light chains of myosin, catalyzed by myosin light chain kinase that is dependent on calcium and calmnodulin. With phosphorylation, there is a coincident increase in the actin-activated myosin NTPase activity, cross bridge interaction and contractile activity. However, this myosin phosphorylation mechanism may not be the sole factor controlling actin-myosin interaction in vascular smooth muscle. Other mechanisms may function in addition to this myosin-linked regulation. A calcium-insensitive regulation of contraction was observed in helical strips of chemically skinned (Triton X-100) arterial smooth muscle. Millimolar concentrations of inosine triphosphate and uridine triphosphate supported concentration dependent force development in the absence of calcium. Force development was a function of the MgNTP concentration. At high free calcium concentrations, an additional component of force was observed. ITP and UTP, in contrast to ATP, are less effective substrates for the myosin light chain kinase, and their effect on actin-myosin interaction is thus less than that of ATP. They are, however, utilized by the myosin NTPase after treatment by ATP-γ-S. The efficacy of the substrate for the activated NTPase is greater for UTP than ITP than for ATP.

Properties of membrane-bound ATPase of some acidophilic heterotrophic bacteria

Membrane-bound ATPase (EC 3.6.1.4) of acidophilic heterotrophic bacteria from mine environment was isolated and characterized. The enzyme preparations fromAcidiphilium symbioticum KM2 and the strains GS18h and GS19h have a pH optimum of 7.7, 8.2 and 7.7, respectively, in the presence of Mg2+ which is required for activity. In an assay system containing Mn2+ or Ca2+ only, some activity was also evident. These enzymes hydrolyzed inorganic diphosphate (PPi), guanosine triphosphate (GTP) and inosine triphosphate (ITP) as the better substrate than ATP and theK m values of the enzymes with respect to ATP were determined to be 238, 157 and 228 μmol/L forA. symbioticum KM2 and the strains GS18h and GS19h, respectively. The activity was stimulated by sulfite while Zn2+, Hg2+, 4-chloromercuribenzoic acid (p-CMB) and the specific inhibitors of F0F1 type ATPase,viz. N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin and azide reduced the activity of the enzyme preparations.

Effect of nucleotides on the cytosolic free calcium activity and inositol phosphate formation in human glomerular epithelial cells.

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 1. Three-strand exchange reaction.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.

Heat production in chemically skinned smooth muscle of guinea‐pig taenia coli.

1. The rate of heat production of chemically skinned guinea-pig taenia coli smooth muscle at 25 degrees C was measured using microcalorimetric techniques. 2. Muscle strips were mounted isometrically and incubated in solutions containing MgATP (3.2 mM) and phosphocreatine (PCr, 12 mM), pH 6.9. Activation was obtained by the injection of Ca2+ into the sample compartment of the calorimeter. 3. The heat production rate of the resting preparation (pCa 9) was 0.40 +/- 0.03 mW g-1 wet weight (n = 23). During maximal activation (pCa 4.8) the heat rate increased to 1.12 +/- 0.07 mW g-1 (mean +/- S.E.M., n = 15). With stepwise increase in [Ca2+] from pCa 9 to 4.8 the energetic cost of force maintenance tended to increase at higher [Ca2+]. 4. After activation by Ca2+, the heat production rate reached its maximum while force was still increasing. 5. Changing ionic strength from 90 to 150 mM had no effect on either basal or activated heat rate. Oligomycin, amphotericin B and the adenylate kinase inhibitor Ap5A had no effect on the basal heat rate. 6. Exchanging ATP in the incubation medium for inosine triphosphate (ITP) reduced the force and heat production after injection of Ca2+. The basal heat production was not lowered when ATP was exchanged for ITP. 7. The observed enthalpy change for PCr splitting at 25 degrees C (pH 6.9, ionic strength 90 mM) was -28 +/- 3 kJ mol-1 (mean +/- S.E.M., n = 9). After correction for the phosphate equilibrium, buffer reactions, and Mg2+ binding to PCr and HPO42-, the net enthalpy change is calculated to be -39 +/- 3 kJ mol-1. 8. Heat production in the skinned smooth muscle consists of one basal component present in relaxed muscle, and one component associated with contraction. The nature of the basal heat production is unclear but does not seem to involve turnover of phosphate on the myosin light chains. The increase in the energetic tension cost with increasing activation by Ca2+ has implications for the understanding of the contractile mechanism in smooth muscle.