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The role of endogenous nitric oxide (NO) in the growth and vascularization of a rat carcinosarcoma (P22) has been investigated. Tumor-bearing animals were treated with (i) nitric oxide synthase (NOS) inhibitors, administered via the drinking water, including N(G)-nitro-l-arginine methyl ester (L-NAME), a nonisoform-selective inhibitor, and 2 others that target the inducible (NOS II) enzyme preferentially, namely 1-amino-2-hydroxyguanidine or N-[3-(aminomethyl)benzyl]acetamidine hydrochloride; or (ii) daily injections (intraperitoneally) of 2 Ru(III) polyaminocarboxylates, AMD6221 and AMD6245, both of which are effective NO scavengers. L-NAME, AMD6221, and AMD6245 reduced tumor growth by approximately 60% to 75% of control rates. Tumor sections stained with abs to CD-31/platelet endothelial cell adhesion molecule-1 or NOS III showed that this was associated with a marked reduction (60%-77%) of tumor microvascular densitiy (MVD). Tumors resumed growing promptly when treatment was discontinued, accompanied by partial or complete restoration of MVDs. In contrast, NOS-II selective inhibitors had no effect on tumor growth or vascularization, indicating that both responses require complete blockade of NO production. The results corroborate the view that endogenous NO facilitates tumor development. We suggest that NO deprivation causes tumor feeder vessels to constrict, reducing tumor blood flow. The delivery of oxygen and essential nutrients to the developing tumor is impaired as a consequence, hampering further growth. Normalizing NO levels by withholding treatment causes tumor feeder vessels to dilate, increasing tumor perfusion and reestablishing conditions that allow tumors to begin growing again.

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires beta3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack beta3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing beta3 integrin (CHOwtbeta3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the beta3 integrin cytoplasmic tail. Moreover, CHOwtbeta3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated beta3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

Intermediate filament (IF) proteins exist in multiple structural forms within cells including mature IF, short filaments or 'squiggles', and non-filamentous precursors called particles. These forms are interconvertible and their relative abundance is IF type, cell type- and cell cycle stage-dependent. These structures are often associated with molecular motors, such as kinesin and dynein, and are therefore capable of translocating through the cytoplasm along microtubules. The assembly of mature IF from their precursor particles is also coupled to translation. These dynamic properties of IF provide mechanisms for regulating their reorganization and assembly in response to the functional requirements of cells. The recent findings that IF and their precursors are frequently associated with signaling molecules have revealed new functions for IF beyond their more traditional roles as mechanical integrators of cells and tissues.

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-kappaB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-kappaB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IkappaBalpha and IkappaBbeta1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3kappaB conA-luciferase reporter was also increased, confirming that NF-kappaB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-kappaB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IkappaB and that induction by flow does not involve TPL-2 kinase or PKC.

Photorelaxation of vascular smooth muscle (VSM) was studied using segments of tail artery from normotensive rats (NTR) or spontaneously hypertensive rats (SHR). Isolated vessels with intact endothelium were perfused with Krebs solution containing phenylephrine. Perfusion pressures were recorded while arteries were irradiated with either visible (VIS; lambda=514.5 nm) or long wavelength ultra-violet (UVA; lambda=366 nm) light. VIS light produced a transient vasodilator response: a rapid decrease of pressure that recovered fully during the period (6 min) of illumination. An irradiated artery was refractory to a second period of illumination delivered immediately after the first, but its photosensitivity recovered slowly in the dark, a process called 'repriming'. Photorelaxations generated by UVA light were qualitatively different and consisted of two components: a phasic (or p-) component superimposed on a sustained (or s-) component. The p-component is similar to the VIS light-induced response in that both exhibit refractoriness and repriming depends upon endothelium-derived NO. In contrast, the s-component persists throughout the period of illumination and does not show refractoriness. We conclude that VIS light-induced photorelaxations and the p-component of UVA light-induced responses are mediated by the photochemical release of NO from a finite molecular 'store' that can be reconstituted afterwards in the dark. The s-component of the UVA light-induced response does not depend directly on endothelial NO and may result instead from a stimulatory effect of UVA light on soluble guanylate cyclase. NO-dependent photorelaxation is impaired in vessels from SHR while the s-component is enhanced.

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a finite molecular store that can be depleted by irradiating pre-contracted arteries with visible light. The ability of an 'exhausted' vessel to respond to a further period of illumination is lost temporarily but then recovers slowly as the photosensitive store is reconstituted in the dark. The recovery process, termed repriming, displays an absolute requirement for endothelium-derived NO and is inhibited by pre-treating arteries with ethacrynic acid, a thiol-alkylating agent. Here we demonstrate that agents that up- or down-regulate glutathione (GSH) biosynthesis influence the extent to which the store is regenerated in the dark. Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE] +/- s.e.mean: 5. 78+/-0.46 microM) and periodically exposed to laser light (lambda=514.5 nm, 6.3 mW cm(-2) for 6 min). Photorelaxations of control RTAs were compared with those from either (a) vessels taken from animals previously injected i.p. with buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (three injections, 100 mg kg(-1) at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine (10(-4) M OXO for 60 min). RTAs from BSO-treated animals exhibited attenuated photorelaxations: the mean (+/-s.e.mean) amplitude of the response recorded after 72 min recovery in the dark was 12.4+/-1.6% versus 21.4+/-2.9% for control arteries (n=5; P<0. 01). Conversely RTAs treated with OXO and allowed to recover for a similar period showed enhanced photorelaxations, 32.6+/-6.3% as compared to 21.4+/-2.9% for control arteries (n=5; P<0.01). A hyperbolic curve fit to repriming curves for BSO-treated and control arteries returned asymptote values (maximum photorelaxations) of (mean +/- s.e.mean) 24.2+/-3.2% and 55.2+/-8.5%, respectively. The level of GSH in RTA extracts was measured by high-pressure liquid chromatography (HPLC). Injecting animals with BSO decreased GSH to 85% of control levels (P<0.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P<0.05). Thus, drug-induced changes in RTA GSH levels were positively correlated with altered photorelaxations. The results lead us to postulate that the photosensitive store in VSM is generated, at least in part, from intracellular GSH which becomes converted to S-nitrosoglutathione (GSNO) by nitrosating species that are formed ultimately from endothelium-derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.

A number of amino acid methyl esters have been coupled to N-acetylpenicillamine to give a range of sulfur-containing dipeptides. These have been nitrosated to give a family of structurally related NO-donor drugs. The catalytic effect of copper ions upon the release of NO from these compounds is much less than that upon S-nitroso-N-acetylpenicillamine. However, all the nitrosated dipeptides respond in a similar way with little variation in the value of kCu. On the other hand, the vasodilator action of these compounds and the inhibiting effect of hemoglobin do vary quite considerably within the family. It is suggested that this indicates some tissue penetration by these drugs.

1. Ruthenium(III) reacts with nitric oxide (NO) to form stable ruthenium(II) mononitrosyls. Several Ru(III) complexes were synthesized and a study made of their ability to bind NO, in vitro and also in several biological systems following expression of the inducible isoform of nitric oxide synthase (iNOS). Here we report on the properties of two, related polyaminocarboxylate-ruthenium complexes: potassium chloro[hydrogen(ethylenedinitrilo)tetraacetato]ruthenate+ ++ (=JM1226; CAS no.14741-19-6) and aqua[hydrogen(ethylenedinitrilo)tetraacetato]ruthenium (=JM6245; CAS no.15282-93-6). 2. Binding of authentic NO by aqueous solutions of JM1226 yielded a product with an infrared (IR) spectrum characteristic of an Ru(II)-NO adduct. A compound with a similar IR spectrum was obtained after reacting JM1226 with S-nitroso-N-acetylpenicillamine (SNAP). 3. The effect of JM1226 or JM6245 on nitrite (NO2-) accumulation in cultures of macrophages (RAW 264 line) 18 h after stimulating cells with lipolysaccharide (LPS) and interferon-gamma (IFNgamma) was studied. Activation of RAW264 cells increased NO2- levels in the growth medium from (mean+/-1 s.e.mean) 4.9+/-0.5 microM to 20.9+/-0.4 microM. This was blocked by actinomycin D (10 microM) or cycloheximide (5 microM). The addition of JM1226 or JM6245 (both 100 microM) to activated RAW264 cells reduced NO2- levels to 7.6+/-0.2 microM and 8.8+/-0.6 microM, respectively. N(G)-methyl-L-arginine (L-NMMA; 250 microM) similarly reduced NO2- levels, to 6.1+/-0.2 microM. 4. The effect of JM1226 or JM6245 on NO-mediated tumour cell killing by LPS+IFNgamma-activated macrophages (RAW 264) was studied in a co-culture system, using a non-adherent murine mastocytoma (P815) line as the 'target' cell. Addition of JM1226 or JM6245 (both 100 microM) to the culture medium afforded some protection from macrophage-mediated cell killing: target cell viability increased from 54.5+/-3.3% to 93.2+/-7.1% and 80.0+/-4.6%, respectively (n=6). 5. Vasodilator responses of isolated, perfused, pre-contracted rat tail arteries elicited by bolus injections (10 microl) of SNAP were attenuated by the addition of JM1226 or JM6245 (10(-4) M) to the perfusate: the ED50 increased from 6.0 microM (Krebs only) to 1.8 mM (Krebs + JM6245) and from 7 microM (Krebs only) to 132 microM (Krebs + JM1226). Oxyhaemoglobin (5 microM) increased the ED50 value for SNAP from 8 microM to 200 microM. 6. Male Wistar rats were injected with bacterial LPS (4 mg kg(-1); i.p.) to induce endotoxaemia. JM1226 and JM6245 (both 100 microM) fully reversed the hyporesponsiveness to phenylephrine of tail arteries isolated from animals previously (24 h earlier) injected with LPS. Blood pressure recordings were made in conscious LPS-treated rats using a tail cuff apparatus. A single injection of JM1226 (100 mg kg(-1), i.p.) administered 20 h after LPS (4 mg kg(-1), i.p.) reversed the hypotension associated with endotoxaemia. 7. The results show that JM1226 and JM6245 are able to scavenge NO in biological systems and suggest a role for these compounds in novel therapeutic strategies aimed at alleviating NO-mediated disease states.

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO). 2. Bolus injections (10 microl) of SNAP or GSNO (10(-7)-10(-3) M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1-7 microM) to develop pressures of 100-120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10(-4) M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10(-5) M) and then injected with either pure SNAP or GSNO. 3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.

A number of S-nitrosated compounds derived from 1-thiosugars (glucose, galactose, xylose, maltose, and lactose) have been prepared and characterized. Most of the compounds obtained were unstable either as solids or in solution. However, S-nitroso-1-thio-2,3,4,6-tetra-O-acetylglucopyranose was stable enough to examine as a vasodilator using an isolated rat tail artery model. It also proved effective in human cutaneous vascular smooth muscle relaxation when delivered transdermally.

1. The vasodilator properties and photochemical decomposition of two synthetic iron-sulphur-nitrosyl clusters (cluster A: [Fe4S4(NO)4], tetranitrosyl-tetra-mu 3-sulphido-tetrahedro-tetrairon; and B:[Fe4S3 (NO)7]-1, heptanitrosyl-tri-mu 3-thioxotetraferrate(-1)) have been investigated. Experiments were carried out on isolated, internally-perfused segments of rat tail artery. 2. Bolus injections (10 microliters) of A or B ( > 0.25 mM) delivered into the internal perfusate generated sustained (or S-type) vasodilator responses, characterized by a persistent plateau of reduced tone due to NO released from clusters which enter and become trapped within endothelial cells. Clusters were therefore irradiated with visible laser light (lambda = 457.9 or 514.5 nm) either (a) in solution, while passing through a glass tube en route to the artery; or (b) when retained within the endothelium, by illuminating the artery directly during the plateau of an S-type response. Irradiation produced an additional vasodilator response, the magnitude of which depended upon wavelength and laser beam energy. 3. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM), had no effect on light-induced vasodilator responses. However, they were (a) blocked entirely by adding oxyhaemoglobin (5 microM) to the internal perfusate; and (b) greatly enhanced by the enzyme superoxide dismutase (150 u ml-1). 4. Photolysis of cluster B was measured by absorption spectroscopy and by detecting NO released with an electrochemical sensor. The photochemical reaction was found to be oxygen-dependent. The half-time for inactivation of cluster-derived NO was measured by interposing different lengths of tubing (i.e. time delays) between the photolysis tube and NO sensor. The steady-state probe current decayed exponentially with increasing delay time, with a t 1/2 of 21 s. The amplitudes of vasodilator responses of the tail artery also decreased exponentially by increasing the time delay (t 1/2 = 58 s). Superoxide dismutase (150 u ml-1) prevented this from happening, showing that "inactivation' of cluster-derived NO was caused by reaction with superoxide anions formed during photolysis. 5. We conclude that potentiation of vasodilator responses to iron-sulphur-nitrosyl clusters by visible light is due to an oxygen-dependent photochemical reaction which accelerates the release of ligated nitrosyl groups as free NO. Based on our measurements, we estimate that ca 100 pM NO is sufficient to produce a just-detectable additional vasodilatation and that the ED50 dose is ca 3.7 nM.

The chemistries of S-nitroso-DL-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) in relation to their ability to relax vascular smooth muscle and prevent platelet aggregation have been investigated. Metal ion catalysis greatly accelerates the decomposition of SNAP, but has little effect on GSNO. Instead, NO release from GSNO is effected either by NO transfer to a free thiol (e.g. cysteine), or by enzymatic cleavage of the glutamyl-cystyl peptide bond. In both cases the resulting nitrosothiol (i.e. S-nitrosocysteine and S-nitrosocystylglycine, respectively) is susceptible to metal ion catalysed NO release. We conclude that transnitrosation or enzymatic cleavage are obligatory steps in the mechanism of NO release from GSNO, whereas SNAP needs only the presence of metal ions to effect this process. The different modes of NO production may go some way towards explaining the different physiological effectiveness of these S-nitrosothiols as vasodilators and inhibitors of platelet aggregation.

The multiplicity of biological functions thus far attributed to NO has led to suggestions that some effects might be mediated by other, related species instead. The radical nature of NO cannot account for its cytotoxicity, but its reaction with superoxide to form peroxynitite and highly reactive hydroxyl radicals may be important in this context. The ease with which NO can react with and destroy Fe-S clusters is also an important factor. Nitrosonium and nitroxide ions can be produced in vivo and will react under conditions that are physiologically relevant. Both could, in theory, serve in cell signalling or as cytotoxic agents. More direct experimental evidence for their involvement is needed before we can confidently assign them specific biological roles. In this article, Anthony Butler, Frederick Flitney and Lyn Williams discuss the chemistry of NO and related species.

Nicotinamide (NA) is currently entering clinical trials as a radiosensitizer. A major component of its activity is the improvement of tumour oxygenation resulting from a reduction in microregional ischaemia. NA is known to reduce arterial blood pressure in rodents, suggesting a vascular component in its mechanism of action. We have used an ex vivo system to study the direct action of NA on the contractile properties of vascular smooth muscle. Isolated pieces of rat tail artery were internally perfused with Krebs' solution at a constant flow rate so that constriction of the arterial smooth muscle could be measured as an increase in perfusion pressure. Transient vasoconstrictor responses lasting up to 10 min were induced with bolus injections (10 microliters) of phenylephrine, at concentrations ranging from 10(-5) to 10(-2) M, into the internal perfusate whereas a constant increase in vasoconstrictor tone, giving perfusion pressures of 43-84 mmHg, was induced by constantly perfusing with PE (5 x 10(-6) M) or raising the K+ concentration of the Krebs' solution to 122 mM. The addition of NA to the perfusate significantly reduced the size of the transient vasoconstrictor responses in a dose-dependent manner and induced the precontracted vessels to relax. This action of NA could not be blocked either by N omega-nitro-L-arginine methyl ester (L-NAME), indomethacin or propranolol. We conclude that direct effects on supplying blood vessels probably contribute to the oxygenating action of NA in tumours, though the precise mechanism remains obscure.

1. Two iron-sulphur cluster nitrosyls have been investigated as potential nitric oxide (NO.) donor drugs (A: tetranitrosyltetra-mu 3-sulphidotetrahedro-tetrairon; and B: heptanitrosyltri-mu 3-thioxotetraferrate(1-)). Both compounds are shown to dilate precontracted, internally-perfused rat tail arteries. 2. Bolus injections (10 microliters) of compound A or B generate two kinds of vasodilator response. Doses below a critical threshold concentration (DT) evoke transient (or T-type) responses, which resemble those seen with conventional nitrovasodilators. Doses > DT produce sustained (or S-type) responses, comprising an initial, rapid drop of pressure, followed by incomplete recovery, resulting in a plateau of reduced tone which can persist for several hours. 3. T- and S-type responses are attenuated by ferrohaemoglobin (Hb) and by methylene blue (MB), but not by inhibitors of endothelial NO. synthase. Addition of either Hb or MB to the internal perfusate can restore agonist-induced tone when administered during the plateau phase of an S-type response. Moreover, subsequent removal of Hb causes the artery to re-dilate fully. 4. We conclude that T- and S-type responses are both mediated by NO.. It is postulated that S-type responses represent the sum of two vasodilator components: a reversible component, superimposed upon a non-recoverable component. The former is attributed to free NO., preformed in solution at the time of injection; and the latter to NO. generated by gradual decomposition of a 'store' of iron-sulphur-nitrosyl complexes within the tissue. This hypothesis is supported by histochemical studies which show that both clusters accumulate in endothelial cells.

1. A study has been made of the decline in contractility and some associated metabolic changes which occur in the isolated frog ventricle during the development of hypodynamic depression. 2. The release of two identified prostaglandins (PG), E1 and E2, together with several as yet unknown prostaglandin-related substances (PRS), accompanies the development of hypodynamic depression. There is a close correlation between the extent to which the isometric twitch is depressed and the quantity of prostaglandin released into the superfusate. 3. Fractionation of extracts of 'used' superfusates, using preparative-scale thin-layer chromatography, revealed the presence of six major components, four of which (PGE1 and PGE2 and two unidentified components) were found to be cardioactive and potentiated contraction when tested subsequently on hypodynamic preparations. 4. Two agents which influence prostaglandin biosynthesis, arachidonic acid and indomethacin, are found to affect both the rate at which the hypodynamic state develops and the extent to which the 'steady-state' twitch tension is depressed, in a dose-dependent manner. Indomethacin, a PG-synthetase inhibitor, accelerates the decay and depresses the final 'steady-state' tension attained, whereas arachidonic acid, the principal precursor for prostaglandin biosynthesis, has the converse effects. 5. Measurements of endogenous 3'5'-cyclic nucleotide levels reveal a time-dependent decrease in intracellular adenosine 3'5'-cyclic monophosphate (3'5'-cyclic AMP) and a concomitant increase in guanosine 3'5' cyclic monophosphate (3'5'-cyclic GMP). The decline in isometric twitch tension is paralleled almost exactly by an equivalent reduction in the ratio 3'5'-cyclic AMP: 3'5'-cyclic GMP. 6. Superfusion of isolated ventricles with Ringer solution containing exogenous, lipid-soluble derivatives of 3'5'-cyclic AMP and 3'5'-cyclic GMP affects both the rate of decline of the isometric twitch and the steady-state tension ultimately reached: thus, 8-bromo-3'5'-cyclic GMP accelerates the decline in contractility and depresses the steady-state level, whereas dibutyryl 3'5'-cyclic AMP delays the development of hypodynamic depression, and elevates the final twitch tension. The effects of both 3'5' cyclic nucleotide derivatives are dose-dependent. 7. The possible involvement of prostaglandins and 3'5'-cyclic nucleotides as causal agents in the mechanism of hypodynamic depression is discussed. The biochemical basis for the implied antangonistic effects of 3'5'-cyclic AMP and 3'5'-cyclic GMP in regulating ventricular contractility is considered in the following paper (Flitney & Singh, 1980).

A study has been made of the effects of uridine 5'-triphosphate (UTP) on the isolated frog ventricle. Preparations were superfused with solutions containing different concentrations of UTP, and changes in contractility, cyclic nucleotide levels and membrane potential were measured. UTP produced a long lasting increase in isometric twitch tension, which was unaffected by adrenergic receptor antagonists (propranolol and phentolamine). The levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of the ventricle to 10(-4) M UTP. The increase in the force of contraction was found to be accompanied by a rise in intracellular cyclic AMP. Cyclic GMP levels were seen to fall initially, but then to increase later, as both twitch and cyclic AMP started to decline. UTP also produced marked changes in the shape of the action potential; its duration and positive overshoot were both increased. The effects of UTP on twitch tension, cyclic nucleotide levels and action potential parameters were all dose-related. The change in contractility was found to be linearly related to (a) the ratio of the amount of cyclic AMP: cyclic GMP present in the fibres, and (b) to the increase in duration of the action potential. These results suggest that cyclic AMP, cyclic GMP and the availability of Ca2+ may all be involved in mediating the response to UTP.

1. A study has been made of the tension responses and sarcomere length changes produced by servo-controlled stretches applied to isometrically contracting frog muscle. Sarcomere lengths were monitored by cine-photography of diffiraction spectra obtained by illuminating a small area of muscle with a laser. 2. The tension increment produced by a ramp-and-hold stretch of approximately 1 mm (ca. 4% of the muscle length) comprises three phases whose limits are defined by two points, S1 and S2, where the slope of the response decreases abruptly. S1 and S2 correspond to extensions of 0.13 and 1.2% of the muscle length. 3. Movements of the first order spectra relative to the zero order recorded during stretch reveal that S2 coincides with an abrupt elongation of the sarcomeres. This is termed sarcomere 'give' and it occurs when the filaments are displaced by 11-12 nm from their steady-state (isometric) position. 4. The stiffness of the sarcomeres, Es, up to S2 decreases with increasing sarcomere length. The maximum force sustained by the muscle at S2, PS2, also shows an inverse dependence on sarcomere length. Both Es and PS2 fall to zero at an extrapolated sarcomere spacing of 3.6-3.7 micrometer, coinciding with the length at which the actin and myosin filaments no longer overlap. 5. The ratio PS2/P0 (where P0 = maximum isometric tension) varies with temperature and speed of stretch. It increases with increasing speeds of stretch until a certain critical velocity, Vc, is reached, beyond which it is almost independent of any further increase. Vc has a positive temperature coefficient, increasing 5-6 in the range 0-30 degrees C (Q10 = 1.8). There is a positive correlation between the maximum speed of isotonic shortening (Vmax.) and Vc in different muscles. 6. Sarcomere 'give' during stretch is considered to be due to forcible detachment of cross-bridges between the actin and myosin filaments. This results in recoil of the extended series elastic elements in the muscle at the expense of the sarcomers. The amount of filament displacement required to induce sarcomere 'give' (11-12 nm) is thought to represent the range of movement over which a cross-bridge can remain attached to actin during a stretch.

1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are complex, but the accompanying change in isometric twitch tension is paralleled closely by corresponding changes in the ratio 3',5'cyclic AMP:3',5'-cyclic GMP. 5. It is concluded that ATP exerts a dual effect on the ventricle and that the contractile response is regulated by changes in the metabolism of 3',5'-cyclic nucleotides. The effects of indomethacin indicate a possible involvement of prostaglandins in mediating the ATP response. It is suggested that the initial effect of ATP on the ventricle is to increase the permeability of the fibres to Ca2+. 6. The relationship between 3',5' cyclic nucleotide levels and ventricular contractility is discussed. It is postulated that the antagonistic effects of 3',5'-cyclic AMP and 3',5'-cyclic GMP are expressed at the level of certain phosphoproteins which regulate both the availability of Ca2+ and the sensitivity of the contractile proteins to Ca2+.

1. A study has been made of changes in sarcomere length and tension which occur during relaxation from isometric (;fixed ends') tetani in isolated muscle fibres of the frog. Sarcomere lengths were calculated from measurements of the separation of the zero-to-first-order intensity maxima in diffraction patterns generated by illuminating small segments of fibre with a He-Ne laser. Diffraction spectra were recorded continuously on cine-film using the method of ;streak' photography.2. Many sarcomeres in a muscle fibre are found to undergo active shortening during relaxation, at the expense of others located elsewhere which become passively extended. The time of onset of changes in sarcomere length coincides with the well known ;shoulder' on the tension record, and their amplitude is maximal at the time when isometric force approaches zero. The original pattern of sarcomere lengths is only re-established after tension has disappeared.3. The variability in the pattern of sarcomere length changes during relaxation was studied in a sample of twenty fibres, by making ;streak' recordings from successive 1 mm segments along the entire length of each fibre. This survey showed that segments which elongate are located predominantly, though not exclusively, close to the fibre ends. The fractional length of the fibre which underwent shortening was found to vary considerably in different preparations. In many ;streak' recordings the first order line fragmented into several distinct intensity maxima.4. The effects of varying the initial fibre length on the time course of the tension decay and on the accompanying changes in sarcomere length were studied. The range of fibre lengths investigated corresponded with sarcomere spacings of 1.9-3.2 mum. The rate constant, R, for the exponential (later) component of the tension decay decreased with increasing fibre length and this was accompanied by a reduction in the mean amplitude of sarcomere shortening [Formula: see text] and lengthening [Formula: see text]. However, the time interval during which sarcomeres shortened or elongated increased, and so the mean velocities of sarcomere length changes ([Formula: see text] and [Formula: see text]) also displayed an inverse dependence upon fibre length.5. The effects of altering temperature (0-20 degrees C) on the time course of the changes in sarcomere length and tension were investigated. The time to onset of the length changes, t(1), the time to maximum amplitude, t(2), and the time to full recovery, t(3), all decreased exponentially with increasing temperature, but to differing extents. Temperature co-efficients for the velocity of sarcomere length changes during the interval t(2) - t(1) and for the fast tension decay were 2.47 ([Formula: see text]), 2.54 ([Formula: see text]) and 2.45 (R). The rate of the slow tension decay (before the tension ;shoulder') also increased with temperature, with a Q(10) of 2.43.6. The complex patterns of sarcomere length changes seen during relaxation appear to be due to variations in the duration of mechanical activity in different fibre segments. A model is presented which shows that the characteristic form of the tension decay can be accounted for by the progressive emergence of local inequalities in the relative strengths of adjacent sarcomeres, as relaxation proceeds.

1. The effects of single and double cycles of stretch and release on the tension response and relative sliding movement of the actin and myosin filaments in active frog's muscle were investigated. 2. The cross-bridges linking the filaments together are able to accommodate a greater range of filament displacement before becoming detached during a second cycle stretch, providing it commences without delay following the preceding release: sarcomere 'give' then occurs for displacements of around 18 nm, as compared with 12 nm for a first cycle stretch. It is postulated that the difference arises because the myosin heads adopt different 'preferred' positions in the isometric steady-state and at the end of a previous release. 3. Muscle length-tension loops were recorded and used to measure the energy absorbed when a muscle is subjected to cycles of stretch and release. The work absorbed per unit length change increases with increasing displacement of the cross-bridges from their initial (isometric) steady-state position, up to the point at which sarcomere 'give' occurs (S2); thereafter it remains constant. 4. More work is absorbed during the first cycle of a double stretch-release combination than during the second. The greater amount absorbed during the first cycle is associated with a correspondingly greater amount of filament sliding in the period following sarcomere 'give'. Sarcomere length-tension loops were constructed and these showed that not less than 80-85% of the work done on a muscle is absorbed by the sarcomeres themselves. 5. A greater amount of work is done on stretching up to (but not beyond) S2 during second cycle stretch as compared to a first. The difference amounts about 1 mJ.m-2 per half-sarcomere. 6. The results are compatible with the mechanism for force production proposed by Huxley & Simmons (1973), in which each myosin head generates force in a number of stepping movements, from one attached state to another. It is concluded that (a) during an unloaded isotonic contraction the working 'stroke' of the head would result in a 10-13 nm relative sliding movement of the filaments, and (b) the potential energy difference separating the two 'preferred' states is 6-9.6 kT per cross-bridge, or 3-4.8 kT per S-1 sub-units, assuming that each one interacts simultaneously with the actin filament.

A study has been made of the effects of acetylcholine and adrenaline on the metabolism of 3',5'-cyclic nucleotides by the isolated frog ventricle. Measurements of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were made: (a) at various times during responses produced by a given concn of acetylcholine (10(-7) M) or adrenaline (10(-6) M); and (b) at fixed times following exposure to different concns of the two agonists. The negative inotropic effect of acetylcholine is accompanied by reciprocal changes in the two cyclic nucleotides: cyclic GMP levels rise and cyclic AMP falls. Both effects are dose-related. A more complex pattern of changes occurs during responses produced by adrenaline. The initial increase in twitch tension is accompanied by a transient reduction in cyclic GMP and by a large increase in cyclic AMP. The rate at which cyclic AMP accumulates is greatest at the time when cyclic GMP levels are maximally depressed. An increase in cyclic GMP, seen later, coincides with a gradual fall in both twitch tension and cyclic AMP. The increments in both cyclic AMP and cyclic GMP, measured when the twitch is maximally potentiated, are dose-related. The magnitude of the decrease in peak twitch tension produced by acetylcholine and of the increase produced by adrenaline correlate closely with corresponding changes in the relative proportion of cyclic AMP: cyclic GMP present in the fibres. The possible significance of the changes in cyclic AMP and cyclic GMP in modulating the response of the ventricle is discussed.

1. A study has been made of some optical and associated mechanical properties of resting frog's sartorius muscles in isotonic and hypertonic solutions. Tension and transparency changes accompanying small alterations of muscle length (smaller than 1-5%) were recorded simultaneously. 2. The form of the transparency change is complex. It has three phases, two of which occur during the length change and the third (delayed phase) after it is complete. Directional recording of the response reveals both scattering and diffraction components. 3. The change of light scattering is associated with tension changes in the short-range elastic component (SREC) of the muscle. Its magnitude is related to the stiffness of the SREC; both increase when the osmotic strength of the external solution is raised and when muscle length is increased. 4. The change of light scattering is not much affected by the velocity of the applied length change. This is true also of the mechanical stiffness of the SREC. 5. The origin of the diffraction change is not known. 6. It is concluded that the scattering effect is caused by conformational changes in the SREC.

Stretch of active whole muscle causes a sudden yielding of the sarcomeres when the actin and myosin filaments are displaced by 10-12 nm from their isometric position. This behaviour is not normally seen during stretch of singly-dissected fibres, but it can be induced by introducing series compliance into the system.