Comp Physiol Research Articles

Protein Kinase G-I Deficiency Induces Pulmonary Hypertension through Rho A/Rho Kinase Activation

Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.

Rebuttal from Mounier and Brugniaux

Point:CounterpointRebuttal from Mounier and BrugniauxPublished Online:15 May 2012https://doi.org/10.1152/japplphysiol.00067.2012cMoreSectionsPDF (39 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat By arguing for major physiological differences between normobaric (NH) and hypobaric (HH) hypoxia, Millet al. (7) are indirectly pleading for “real altitude” as the gold standard. In our opinion, hypobaric chambers represent nothing more than another (inaccurate?) model, inasmuch as real altitude involves more physiological constraints than simply a reduction in barometric pressure (PB). For instance, Mairbaurl et al. (6) demonstrated that the air dryness occurring at altitude could be misleading in the interpretation of the alterations in the alveolar epithelium.It is interesting to note that Millet et al. (7) also decided to highlight acute mountain sickness and preacclimatization as a point of debate in their argument. However, we have a completely different interpretation of the literature they cited. Essentially, the dose of hypoxia used by Beidleman et al. (1) during their preacclimatization is fundamentally different to what Fulco et al. (4) used (60 h at 4,300 m vs. 525 h at 2,200–3,100 m). We, therefore, argue that more than the changes in PB per se and fewer hours spent at a much lower altitude account for the discrepancy between these two studies. This is a clear illustration of the difficulties in interpreting the available literature, thus warranting further research.On the basis of our original contribution (8), the notion that the erythropoietin response is driven by changes in oxygen pressure (Po2) rather than PB is not subject of debate, which would tend to temper the alleged lack of efficiency of the normobaric living high-training low regimen (nLHTL). As a matter of fact, our group actually demonstrated a clear increase in both O2 carrying capacity and performance in elite athletes using nLHTL (2).Furthermore, the data presented by Millet al. (7) relating to nitric oxide (NO) and fluid balance are not convincing. For instance, the work from Hemmingsson and Linnarsson (5) has triggered heated discussions regarding the validity of the measurements, thus prompting us to the greatest caution regarding the interpretation of their results. Regarding fluid balance, Fall et al. (3) recently demonstrated in NH the role played by exercise and hypoxia on contracting plasma volume, independently of increased diuresis.All in all, although we acknowledge possible marginal differences between HH and NH, we stand by our point arguing that O2 sensing-related physiological adaptations are driven by Po2. However, based on existing gaps in the literature, we think more studies comparing the two models, especially those involving longer exposures, are required.REFERENCES1. Beidleman BA , Muza SR , Fulco CS , Cymerman A , Ditzler D , Stulz D , Staab JE , Skrinar GS , Lewis SF , Sawka MN. Intermittent altitude exposures reduce acute mountain sickness at 4300 m. Clin Sci (Lond) 106: 321–328, 2004.Crossref | PubMed | ISI | Google Scholar2. Brugniaux JV , Schmitt L , Robach P , Nicolet G , Fouillot JP , Moutereau S , Lasne F , Pialoux V , Saas P , Chorvot MC , Cornolo J , Olsen NV , Richalet JP. Eighteen days of “living high, training low” stimulate erythropoiesis and enhance aerobic performance in elite middle-distance runners. J Appl Physiol 100: 203–211, 2006.Link | ISI | Google Scholar3. Fall L , Evans KA , Lewis MH , Bailey DM. Haemostatic response to hypoxaemic/exercise stress: the dilemma of plasma volume correction. J Clin Pathol 64: 269–271, 2011.Crossref | ISI | Google Scholar4. Fulco CS , Muza SR , Beidleman BA , Demes R , Staab JE , Jones JE , Cymerman A. Effect of repeated normobaric hypoxia exposures during sleep on acute mountain sickness, exercise performance, and sleep during exposure to terrestrial altitude. Am J Physiol Regul Integr Comp Physiol 300: R428–R436, 2011.Link | ISI | Google Scholar5. Hemmingsson T , Linnarsson D. Lower exhaled nitric oxide in hypobaric than in normobaric acute hypoxia. Respir Physiol Neurobiol 169: 74–77, 2009.Crossref | ISI | Google Scholar6. Mairbaurl H , Weymann J , Mohrlein A , Swenson ER , Maggiorini M , Gibbs JS , Bartsch P. Nasal epithelium potential difference at high altitude (4,559 m): evidence for secretion. Am J Respir Crit Care Med 167: 862–867, 2003.Crossref | PubMed | ISI | Google Scholar7. Millet GP , Faiss R , Pialoux V. Point: Hypobaric hypoxia induces different physiological responses from normobaric hypoxia. J Appl Physiol; 10.1152/japplphysiol.00067.2012.ISI | Google Scholar8. Mounier R , Brugniaux JV. Counterpoint: Hypobaric hypoxia does not induce different physiological responses from normobaric hypoxia. J Appl Physiol; 10.1152/japplphysiol.00067a.2012.ISI | Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 112Issue 10May 2012Pages 1787-1787 Copyright & PermissionsCopyright © 2012 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00067.2012cPubMed22589491History Published online 15 May 2012 Published in print 15 May 2012 Metrics

Point: Counterpoint: Hypobaric hypoxia induces/does not induce different responses from normobaric hypoxia

Point: Counterpoint: Hypobaric hypoxia induces/does not induce different responses from normobaric hypoxia

Rapid stimulus-bound suppression of intake in response to an intraduodenal nonnutritive sweetener after training with nutritive sugars predicting malaise

In a previous report (Schier et al., Am J Physiol Regul Integr Comp Physiol 301: R1557-R1568, 2011), we demonstrated with a new behavioral procedure that rats exhibit stimulus-bound suppression of intake in response to an intraduodenal (ID) bitter tastant predicting subsequent malaise. With the use of the same modified taste aversion procedure, the present experiments evaluated whether the sweet taste properties of ID stimuli are likewise detected and encoded. Thirsty rats licked at sipper spouts for hypotonic NaCl for 30 min and received brief (first 6 min) yoked ID infusions of either the same NaCl or an isomolar lithium chloride (LiCl) solution in each session. An intestinal taste cue was mixed directly into the LiCl infusate for aversion training. Results showed that rats failed to detect intestinal sweet taste alone (20 mM Sucralose) but clearly suppressed licking in response to a nutritive sweet taste stimulus (234 mM sucrose) in the intestine that had been repeatedly paired with LiCl. Rats trained with ID sucrose in LiCl subsequently generalized responding to ID Sucralose alone at test. Replicating this, rats trained with ID Sucralose in compound with 80 mM Polycose rapidly suppressed licking to the 20 mM Sucralose alone in a later test. Furthermore, ID sweet taste signaling did not support the rapid negative feedback of sucrose or Polycose on intake when their digestion and transport were blocked. Together, these results suggest that other signaling pathways and/or transporters engaged by caloric carbohydrate stimuli potentiate detection of sweet taste signals in the intestine.

Spectral Tuning between Vision and Bioluminescence in Beetles, An Example of von Uexkull's Functional Cycle

Spectral Tuning between Vision and Bioluminescence in Beetles, An Example of von Uexkull's Functional Cycle

Editorial: Functional intracellular renin-angiotensin systems: potential for pathophysiology of disease

the renin-angiotensin system (RAS) is a coordinated hormonal cascade playing a major role in cardiovascular, renal, and adrenal homeostasis ([4][1]). Derangements in the RAS have been implicated in the pathogenesis of many disease states, including hypertension and of target organ damage related to

The curious case of anabolic resistance: old wives' tales or new fables?

the progressive loss of muscle mass, commonly termed sarcopenia, accompanies the process of healthy aging ([27][1]). The underlying basis of this condition, in a very simplistic view, would be at a more advanced age skeletal muscle proteins are being lost because of an imbalance between muscle

Comments on Point:Counterpoint: High altitude is/is not for the birds!

TO THE EDITOR: The debate between Scott et al. (6) and Llanos et al. (4) highlights the remarkable ability of birds and mammals to adapt and survive at high altitudes and cope with reduced oxygen (O2) availability. Scott and colleagues (6) make a compelling argument that birds are far superior to mammals at adapting and thriving in hypoxic environments, both at rest and during exercise. While their argument is supported by comparisons between birds and terrestrial mammals, it ignores marine mammals. Marine mammals such as seals are routinely exposed to and tolerate long bouts of hypoxia during breath-hold diving. For example, elephant seals may dive to depths of nearly 1,600 m and occasionally remain submerged for 2 h (3). Moreover, marine mammals can be exposed to partial pressures of arterial oxygen (PaO2) as low as 12 mmHg during free dives (5), which is less than the 20 mmHg observed in the bar-headed goose (6). Interestingly, a PaO2 of 12 mmHg observed in elephant seals corresponds to arterial saturations and O2 content of only 8% and 2.7 ml O2/dl (5). Thus marine mammals such as the elephant seal demonstrate a remarkable hypoxia tolerance. The integration of several key physiological adaptations including 1) substantially greater myoglobin concentrations compared to other mammals, 2) reductions in metabolism, 3) a greater reliance on aerobic metabolism (i.e., less lactate production), 4) a host of dramatic acute cardiovascular adjustments; and 5) an increased intrinsic cerebral hypoxia tolerance allow marine mammals to thrive in hypoxic environments and enable long deep dives (1–3).

Further support for the alignment of cattle along magnetic field lines: reply to Hert et al.

Hert et al. (J Comp Physiol A, 2011) challenged one part of the study by Begall et al. (PNAS 105:13451–13455, 2008) claiming that they could not replicate the finding of preferential magnetic alignment of cattle recorded in aerial images of Google Earth. However, Hert and co-authors used a different statistical approach and applied the statistics on a sample partly unsuitable to examine magnetic alignment. About 50% of their data represent noise (resolution of the images is too poor to enable unambiguous measurement of the direction of body axes, pastures are on slopes, near settlements or high voltage power-lines, etc.). Moreover, the authors have selected for their analysis only ~ 40% of cattle that were present on the pastures analyzed. Here, we reanalyze all usable data and show that cattle significantly align their body axes in North–South direction on pastures analyzed by Hert and co-authors. This finding thus supports our previous study. In addition, we show by using aerial Google Earth images with good resolution, that the magnetic alignment is more pronounced in resting than in standing cattle.Electronic supplementary materialThe online version of this article (doi:10.1007/s00359-011-0674-1) contains supplementary material, which is available to authorized users.

A novel vascular EET synthase: role of CYP2C7

We demonstrated previously that cytochrome P-450 (CYP) 2C29 is the epoxyeicosatrienoic acid (EET) synthase responsible for the EET-mediated flow/shear stress-induced dilation of vessels of female nitric oxide (NO)-deficient mice (Sun D, Yang YM, Jiang H, Wu H, Ojami C, Kaley G, Huang A. Am J Physiol Regul Integr Comp Physiol 298: R862-R869, 2010). In the present study, we aimed to identify which specific CYP isoform(s) is the source of the synthesis and release of EETs in response to stimulation by shear stress in vessels of rats. Cannulated mesenteric arteries isolated from both sexes of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats were perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress stimulated release of EETs in the perfusate of female (but not male) NO-deficient vessels, associated with an EET-mediated vasodilation, in which 11,12- and 14,15-EET contributed predominantly to the responses. Rat CYP cDNA array screened a total of 32 CYP genes of mesenteric arteries, indicating a significant upregulation of CYP2C7 in female L-NAME-treated rats. Endothelial RNA and protein were extracted from intact single vessels. Expression of CYP2C7 mRNA and protein in pooled extractions of endothelial lysate was identified by PCR and Western blot analyses. Transfection of the vessels with CYP2C7 short interfering RNA eliminated the release of EETs, consequently abolishing the EET-mediated flow-induced dilation; these responses, however, were maintained in vessels transfected with nonsilencing short interfering RNA. Knockdown of endothelial CYP2C7 was confirmed by PCR and Western blot analyses. In conclusion, CYP2C7 is an endothelial EET synthase in the female rat vasculature, by which, in NO deficiency, shear stress stimulates the release of EETs to initiate vasodilation.

A novel facet to consider for the effects of butyrate on its target cells. Focus on “The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein”

short-chain fatty acids (SCFAs), acetate, propionate, and butyrate are produced by bacterial fermentation of dietary fiber in the colonic lumen. In the normal human large intestine, SCFA concentrations range from 70 to 100 mM; these SCFAs account for ∼80% of the anions present in the lumen ([5][1

Reply to Lundberg, Larsen, and Weitzberg

to the editor: The letter of Lundberg et al. ([12][1]) does not relate directly to any of the articles we published in this journal ([1][2], [2][3], [6][4]). Rather, the correspondents make a general point on the potential risks to athletes who might choose to supplement their diet with nitrate or (

Corrigendum

CorrigendaCorrigendumPublished Online:01 Aug 2011https://doi.org/10.1152/ajpregu-zh6-7635-corr.2011Original articleMoreSectionsPDF (62 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Volume 64, November 2008Miller AM, Brestoff JR, Phelps CB, Berk EZ, Reynolds TH IV. Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice. Am J Physiol Regul Integr Comp Physiol 295: , 2008. First published September 3, 2008; doi:10.1152/ajpregu.90428.2008.In preparation for additional insulin tolerance testing in mice, an error was discovered in the calculation used to determine the volume of insulin used in this study. The dose of insulin used for insulin tolerance testing was actually 12.5 U/kg rather than 0.25 U/kg as reported. We regret this error and would like to alert the readers of our article that our results reported should be viewed in the context of the correct amount of insulin used, 12.5 U/kg, and not the published dose of 0.25 U/kg. We regret this unfortunate miscalculation and apologize to the readers of this journal.This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Related ArticlesRapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice 01 Nov 2008American Journal of Physiology-Regulatory, Integrative and Comparative Physiology More from this issue > Volume 301Issue 2August 2011Pages R558-R558 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/ajpregu-zh6-7635-corr.2011History Published online 1 August 2011 Published in print 1 August 2011 Metrics

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension.

Failed heart rate recovery at a critical age in 5-HT-deficient mice exposed to episodic anoxia: implications for SIDS

Mice deficient in the transcription factor Pet-1⁻/⁻ have a ∼70% deficiency of brainstem serotonin [5-hydroxytryptamine (5-HT)] neurons and exhibit spontaneous bradycardias in room air at postnatal day (P)5 and P12 and delayed gasping in response to a single episode of anoxia at P4.5 and P9.5 (Cummings KJ, Li A, Deneris ES, Nattie EE. Am J Physiol Regul Integr Comp Physiol 298: R1333-R1342, 2010; and Erickson JT, Sposato BC. J Appl Physiol 106: 1785-1792, 2009). We hypothesized that at a critical age Pet-1⁻/⁻ mice will fail to autoresuscitate during episodic anoxia, ultimately dying from a failure of gasping to restore heart rate (HR). We exposed P5, P8, and P12 Pet-1⁻/⁻ mice and wild-type littermates (WT) to four 30-s episodes of anoxia (97% N₂-3% CO₂), separated by 5 min of room air. We observed excess mortality in Pet-1⁻/⁻ only at P8: 43% of Pet-1⁻/⁻ animals survived past the third episode of anoxia while ∼95% of WT survived all four episodes (P = 0.004). No deaths occurred at P5 and at P12, and one of six Pet-1⁻/⁻ mice died after the fourth episode, while all WT animals survived. At P8, dying Pet-1⁻/⁻ animals had delayed gasping, recovery of HR, and eupnea after the first two episodes of anoxia (P < 0.001 for each); death ultimately occurred when gasping failed to restore HR. Both high- and low-frequency components of HR variability were abnormally elevated in dying Pet-1⁻/⁻ animals following the first episode of anoxia. Dying P8 Pet-1⁻/⁻ animals had significantly fewer 5-HT neurons in the raphe magnus than surviving animals (P < 0.001). Our data indicate a critical developmental window at which a brainstem 5-HT deficiency increases the risk of death during episodes of anoxia. They may apply to the sudden infant death syndrome, which occurs at a critical age and is associated with 5-HT deficiency.

Corrigendum

CorrigendumCorrigendumPublished Online:01 Jun 2011https://doi.org/10.1152/ajpregu.zh6-7624-corr.2011Original articleMoreSectionsPDF (84 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 64, October 2008van der Kleij H, O'Mahony C, Shanahan F, O'Mahony L, Bienenstock J. Protective effects of Lactobacillus reuteri and Bifidobacterium infantis in murine models for colitis do not involve the vagus nerve. Am J Physiol Regul Integr Comp Physiol 295: , 2008. First published July 30, 2008; doi:10.1152/ajpregu.90434.2008 (http://ajpregu.physiology.org/content/295/4/R1131.full).We have become aware of the fact that our laboratory has wrongly identified a bacterium, which we have referred to in a number of publications as Lactobacillus reuteri. Indeed, we can now verify that this bacterium, used in all of our published experiments, is Lactobacillus rhamnosus that we identified by bar coding and DNA fingerprinting. We have asked an independent laboratory, the Bacteria Collection Laboratory for Microbiology, University of Ghent, Belgium to identify the bacterium, and they have confirmed it to be an L. rhamnosus, using DNA fingerprinting via the AFLP technique. Furthermore, they were unable to assign it to any of the seven intraspecific clusters they have previously reported among 118 strains of this species from around the world (Vancanneyt et al., Appl Environ Microbiol 78: 5376–5383, 2006). Future experiments by our group will refer to this bacterium as L. rhamnosus (JB-1).We sincerely apologize to all our colleagues for any difficulties arising from this error.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesProtective effects of Lactobacillus reuteri and Bifidobacterium infantis in murine models for colitis do not involve the vagus nerve 01 Oct 2008American Journal of Physiology-Regulatory, Integrative and Comparative Physiology More from this issue > Volume 300Issue 6June 2011Pages R1596-R1596 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/ajpregu.zh6-7624-corr.2011History Published online 1 June 2011 Published in print 1 June 2011 Metrics

Corrigendum

CorrigendumCorrigendumPublished Online:01 May 2011https://doi.org/10.1152/ajpregu-zh6-7585-corr.2011Original articleMoreSectionsPDF (57 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Volume 69, February 2011Tseng YC, Lee JR, Lee SJ, Hwang PP. Functional analysis of the glucose transporters-1α, -6, and -13.1 expressed by zebrafish epithelial cells. Am J Physiol Regul Integr Comp Physiol 300: . First published December 1, 2010; doi:https://doi.org/10.1152/ajpregu.00144.2010 (http://ajpregu.physiology.org/content/300/2/R321).In the title “Functional analysis of the glucose transporters-1α…” the glucose transporter1-α was misrepresented. The title should be “Functional analysis of the glucose transporters-1a, -6, and -13.1 expressed by zebrafish epithelial cells.”The MO sequences of zglut6 and -13.1 in Table 1 were incorrect. The correct Table 1 follows.Table 1. Morpholino oligonucleotides (MOs) used for the gene translational knockdown assayGene NameMO SequenceStartEndzglut1a5′-ATTGCTTTCCGCCTTCCATCACGTC-3′−6 bp+19 bpzglut65′-GCTTCATCCGAACGGTCTGCCATTG-3′−2 bp+23 bpzglut13.15′-TTCAACTCTAGCGTACGCTCTCTGAT-3′+9 bp+33 bpStandard control5′-CCTCTTACCTCAGTTACAATTTATA-3′This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesFunctional analysis of the glucose transporters-1α, -6, and -13.1 expressed by zebrafish epithelial cells 01 Feb 2011American Journal of Physiology-Regulatory, Integrative and Comparative Physiology More from this issue > Volume 300Issue 5May 2011Pages R1261-R1261 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/ajpregu-zh6-7585-corr.2011History Published online 1 May 2011 Published in print 1 May 2011 Metrics

Connections in chronic kidney disease: connexin 43 and connexin 37 interaction

the prevalence of chronic kidney disease is rising sharply worldwide and affects 13.1% of the population in the US ([3][1]). Patients with chronic kidney disease represent a population not only at risk of progression to end-organ failure but also at higher risk for cardiovascular diseases ([22][2

Invited Commentary

Invited Commentary

Mechanism of ATP loss in nonoxidative contracting muscle

The transition from rest to intense exercise is a challenge to cellular energetics ([11][1], [13][2], [15][3]). The metabolic fuels, i.e., the sources of ATP to sustain muscular contraction, are creatine phosphate and glycogen. Two anaerobic metabolic paths, leading to ATP generation, are catalyzed