The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours.

This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.

Vasopressin regulation of sodium transport in the distal nephron and collecting duct.

Arginine vasopressin (AVP) is released from the posterior pituitary gland during states of hyperosmolality or hypovolemia. AVP is a peptide hormone, with antidiuretic and antinatriuretic properties. It allows the kidneys to increase body water retention predominantly by increasing the cell surface expression of aquaporin water channels in the collecting duct alongside increasing the osmotic driving forces for water reabsorption. The antinatriuretic effects of AVP are mediated by the regulation of sodium transport throughout the distal nephron, from the thick ascending limb through to the collecting duct, which in turn partially facilitates osmotic movement of water. In this review, we will discuss the regulatory role of AVP in sodium transport and summarize the effects of AVP on various molecular targets, including the sodium-potassium-chloride cotransporter NKCC2, the thiazide-sensitive sodium-chloride cotransporter NCC, and the epithelial sodium channel ENaC.

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC.

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antibodies targeting pS124-NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment-specific markers localized pS124-NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124-NCC co-localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich-Wistar rats or vasopressin-deficient Brattleboro rats with the vasopressin type 2 receptor-specific agonist dDAVP significantly increased pS124-NCC abundance, with no changes in total NCC plasma membrane abundance. pS124-NCC levels also increased in abundance in rats after stimulation of the renin-angiotensin-aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, (36)Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane-associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline-inducible MDCKII-NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC.

Short communication: Effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating Holstein cows.

To test the hypothesis that dietary N concentrations affect gut epithelial urea transport by modifying the expression of urea transporter B (UT-B) and aquaporins (AQP), the mRNA expression and protein abundance of UT-B and AQP3, AQP7, AQP8, and AQP10 were investigated in ruminal papillae from 9 lactating dairy cows. Ruminal papillae were harvested from cows fed low N (12.9% crude protein) and high N (17.1% crude protein) diets in a crossover design with 21-d periods. The mRNA expression was determined by real-time reverse transcription-PCR and protein abundance by immunoblotting. The mRNA expression of UT-B was not affected by dietary treatment, whereas mRNA expression of AQP3, 7, and 10 were greater in the high N compared with the low N fed cows. Using peptide-derived rabbit antibodies to cow AQP3, 7, and 8, immunoblotting revealed bands of approximately 27, 27, and 24 kDa in ruminal papillae, respectively. A peptide-derived chicken antibody to cow UT-B detected a band of approximately 30 to 32 kDa in ruminal papillae. The abundance of UT-B and AQP3 and 7 were not affected by dietary treatment. In contrast, the abundance of AQP8 was greater in high N compared with low N diets. In conclusion, AQP3, 7, and 8 were found to be expressed in bovine rumen papillae. None of the investigated transcripts or proteins correlated to the increased rumen epithelial urea permeability observed with low dietary N concentration.

A plate reader-based method for cell water permeability measurement.

Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH(2)O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH(2)O, by changing buffer d-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.

Vasopressin-dependent short-term regulation of aquaporin 4 expressed in Xenopus oocytes.

Aquaporin 4 (AQP4) is abundantly expressed in the perivascular glial endfeet in the central nervous system (CNS), where it is involved in the exchange of fluids between blood and brain. At this location, AQP4 contributes to the formation and/or the absorption of the brain edema that may arise following pathologies such as brain injuries, brain tumours, and cerebral ischemia. As vasopressin and its G-protein-coupled receptor (V1(a)R) have been shown to affect the outcome of brain edema, we have investigated the regulatory interaction between AQP4 and V1(a)R by heterologous expression in Xenopus laevis oocytes. The water permeability of AQP4/V1(a)R-expressing oocytes was reduced in a vasopressin-dependent manner, as a result of V1(a)R-dependent internalization of AQP4. Vasopressin-dependent internalization was not observed in AQP9/V1(a)R-expressing oocytes. The regulatory interaction between AQP4 and V1(a)R involves protein kinase C (PKC) activation and is reduced upon mutation of Ser(180) on AQP4 to an alanine. Thus, the present study demonstrates at the molecular level a functional link between the vasopressin receptor V1(a)R and AQP4. This functional interaction between AQP4 and V1(a)R may prove to be a potential therapeutic target in the prevention and treatment of brain edema.

Long-term regulation of proximal tubule acid-base transporter abundance by angiotensin II.

In the proximal tubule, angiotensin II (Ang-II) regulates HCO(-)(3) reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na(+)/HCO(-)(3) cotransport and Na(+)/H(+) exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na(+)/HCO(-)(3) cotransporter (NBC1) or the apical membrane type 3 Na(+)/H(+) exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59 +/- 9% of control; P<0.05) and Ang-II infusion increased abundance (130 +/- 7% of control; P<0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52 +/- 7% of control; P<0.05) and BBM (32 +/- 7% of control; P<0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P<0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption.

Genomic organization of the mammalian SLC14a2 urea transporter genes.

Urea transporters encoded by the UT-A gene play fundamental roles in the kidney and possibly other tissues. Knowledge of the genomic organization of the mouse, rat and human UT-A genes has enabled the engineering of transgenic and knockout animals and these have helped refine our understanding of the role of UT-A proteins. This review summarizes the published work that has accrued on the structure and regulation of these genes. It also documents a novel cDNA, human UT-A3, which has enabled a major refinement of the human UT-A gene structure. This and other information contained in this review should prove useful for future comparative genomic analysis, studies addressing gene regulation and for the engineering of transgenic and knockout animal strains.

Role of collecting duct urea transporters in the kidney--insights from mouse models.

Urea movement across plasma membranes is modulated by specialized urea transporter proteins. These proteins are proposed to play key roles in the urinary concentrating mechanism and fluid homeostasis. To date, two urea-transporter genes have been cloned; UT-A (Slc14a2), encoding at least five proteins and UT-B (Slc14a1) encoding a single protein isoform. Recently we engineered mice that lack the inner medullary collecting duct (IMCD) urea transporters, UT-A1 and UT-A3 (UT-A1/3 -/- mice). This article includes 1) a historical review of the role of renal urea transporters in renal function; 2) a review of our studies utilizing the UT-A1/3 -/- mice; 3) description of an additional line of transgenic mice in which beta-galactosidase expression is driven by the alpha-promoter of the UT-A gene, which is allowing better physiological definition of control mechanisms for UT-A expression; and 4) a discussion of the implications of the studies in transgenic mice for the teaching of kidney physiology.

Characterization of mouse urea transporters UT-A1 and UT-A2.

Specialized transporter proteins that are the products of two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1), modulate the movement of urea across cell membranes. The purpose of this study was to characterize the mouse variants of two major products of the UT-A gene, UT-A1 and UT-A2. Screening a mouse kidney inner medulla cDNA library yielded 4,047- and 2,876-bp cDNAs, the mouse homologues of UT-A1 and UT-A2. Northern blot analysis showed high levels of UT-A mRNAs in kidney medulla. UT-A transcripts were also present in testes, heart, brain, and liver. Immunoblots with an antiserum raised to the 19 COOH-terminal amino acids of rat UT-A1 (L194) identified immunoreactive proteins in kidney, testes, heart, brain, and liver and showed a complex pattern of differential expression. Relative to other tissues, kidney and brain had the highest levels of UT-A protein expression. In kidney sections, immunostaining with L194 revealed immunoreactive proteins in type 1 (short) and type 3 (long) thin descending limbs of the loop of Henle and in the middle and terminal inner medullary collecting ducts. Expression in Xenopus laevis oocytes showed that, characteristic of UT-A family members, the cDNAs encoded phloretin-inhibitable urea transporters. Acute application of PKA agonists (cAMP/forskolin/IBMX) caused a significant increase in UT-A1- and UT-A3-, but not UT-A2-mediated, urea transport.

Coordinated expression of UT-A and UT-B urea transporters in rat testis.

The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ~1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II-III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway.

Structure and characterization of the mouse UT-A gene (Slc14a2).

The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A (Slc14a2) and UT-B (Slc14a1). By genomic library screening and P1 artificial chromosome "shotgun" sequencing, we have determined the structure of the mouse UT-A gene. The gene is >300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5'-region of the gene is the UT-Aalpha promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-Abeta, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 microM dibutryl cAMP, 25 microM forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-Aalpha promoter construct 6.2-fold [from 0.026 +/- 0.003 to 0.160 +/- 0.004, relative light units (RLU)/microg protein] and a 2.4-kb UT-Abeta promoter construct 9.5-fold (from 0.020 +/- 0.002 to 0.190 +/- 0.043 RLU/microg protein) above that in untreated controls. Interestingly, only the UT-Abeta promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kg H(2)O with NaCl caused a significant increase (from 0.060 +/- 0.004 to 0.095 +/- 0.010 RLU/microg protein) in UT-Aalpha promoter activity but had no effect on the UT-Abeta promoter. A tonicity-responsive enhancer was identified in UT-Aalpha and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.

Antiadrenergic effects of adenosine in pressure overload hypertrophy.

In the present study, we sought to evaluate whether the antiadrenergic action of adenosine in the heart is altered in pressure overload hypertrophy produced in rats by suprarenal aortic banding. Epicardial and coronary effluent adenosine and inosine concentrations and release were significantly elevated in compensated pressure overload hypertrophy but not in hearts with left ventricular failure. In pressure overload hearts, the contractile response to beta-adrenergic stimulation was less inhibited by incremental concentrations of either adenosine or the selective A(1) receptor agonist chloro-N:(6)-cyclopentyl adenosine than in controls. Furthermore, the extent of desensitization to the antiadrenergic actions of adenosine in pressure overload hypertrophy appeared to be proportional to the extent of chamber dilation and dysfunction. A 60-minute infusion of adenosine produced a sustained antiadrenergic effect that lasted up to 45 minutes after the infusion was terminated in both controls and hearts with compensated hypertrophy. This effect was not observed in the decompensated left ventricular failure group. Subsequent infusion with adenosine of the A(2A) receptor antagonist 8-(3-chlorostyryl)-caffeine to counteract the proadrenergic effect of A(2A) receptor stimulation did not alter the decreased sensitivity to the antiadrenergic actions of adenosine in hypertrophied hearts. Finally, isolated myocytes from hypertrophied hearts demonstrated a decreased ability to suppress isoproterenol-elicited increases in [Ca(2+)](i) transients in the presence of adenosine and the A(2A) receptor antagonist compared with myocytes from control hearts. Myocardial adenosine concentrations increase during the compensated phase of pressure overload hypertrophy but then decrease when there is evidence of decompensation. The antiadrenergic actions of adenosine transduced via the myocardial A(1) receptor are diminished in pressure overload hypertrophied hearts. These factors may render these hearts more vulnerable to the detrimental effects of chronically increased sympathetic activity.

Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis.

Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2. 0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.

Norepinephrine concentrations in the epicardial transudate reflect early changes in adrenergic activity in the isolated perfused heart.

The aim of this study was to establish whether epicardial transudates could be used to uncover small, but physiologically important changes in interstitial NE concentrations under normal and pathological conditions. Norepinephrine (NE) concentrations measured in epicardial transudate fluid were compared to NE levels in the coronary effluent in normal and pressure overload hypertrophied (POH) rat hearts. Hearts were isolated together with the stellate ganglion and perfused in the inverted position. Epicardial surface transudates, representative fluid of the interstitial myocardial compartment, and coronary effluents were collected for determination of NE levels in the presence and absence of stellate ganglion stimulation. The same protocol was repeated in the presence and absence of nisoxetine, a NE uptake blocker. NE concentrations in epicardial transudates were 16- and 19-fold higher than in the coronary effluent in both sham and POH groups, respectively. NE concentrations in the transudates but not in the coronary effluents were significantly higher (1.6-fold) in hearts with POH when compared to normal hearts. Likewise, nisoxetine (10(-5)m) increased (1.3-fold) NE concentrations in the transudates but not in the effluents of sham animals. As expected, stellate ganglion stimulation increased NE concentrations in both transudates and effluents in sham and POH hearts. In conclusion, determination of NE concentrations in epicardial transudates represents a simple, rapid and sensitive method to detect increases in adrenergic activity in normal and abnormal hearts.

Adenosine A(2a)-receptor activation increases contractility in isolated perfused hearts.

Adenosine A(2a)-receptor activation enhances shortening of isolated cardiomyocytes. In the present study the effect of A(2a)-receptor activation on the contractile performance of isolated rat hearts was investigated by recording left ventricular pressure (LVP) and the maximal rate of LVP development (+dP/dt(max)). With constant-pressure perfusion, adenosine caused concentration-dependent increases in LVP and +dP/dt(max), with detectable increases of 4.1 and 4.8% at 10(-6) M and maximal increases of 12.0 and 11.1% at 10(-4) M, respectively. The contractile responses were prevented by the A(2a)-receptor antagonists chlorostyryl-caffeine and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385) but were not affected by the beta(1)-adrenergic antagonist atenolol. The adenosine A(1)-receptor antagonist dipropylcyclopentylxanthine and pertussis toxin potentiated the positive inotropic effects of adenosine. The A(2a)-receptor agonists ethylcarboxamidoadenosine and dimethoxyphenyl-methylphenylethyl-adenosine also enhanced contractility. With constant-flow perfusion, 10(-5) M adenosine increased LVP and +dP/dt(max) by 5.5 and 6.0%, respectively. In the presence of the coronary vasodilator hydralazine, adenosine increased LVP and +dP/dt(max) by 7.5 and 7.4%, respectively. Dipropylcyclopentylxanthine potentiated the adenosine contractile responses with constant-flow perfusion in the absence and presence of hydralazine. These increases in contractile performance were also antagonized by chlorostyryl-caffeine and ZM-241385. The results indicate that adenosine increases contractile performance via activation of A(2a) receptors in the intact heart independent of beta(1)-adrenergic receptor activation or changes in coronary flow.

Aging reduces the cardioprotective effect of ischemic preconditioning in the rat heart.

Multiple brief periods of ischemia in the mammalian heart elicits protection against morphologic and functional damage caused by longer-duration ischemia. Preconditioning-induced protection against post-ischemic contractile dysfunction has been reported to be depressed with aging of the adult heart. This study was undertaken to determine whether aging of the adult myocardium reduces the preconditioning-induced attenuation of necrosis observed with ischemia. Isolated, perfused hearts obtained from Fischer 344 rats of either 3 (young) or 22 (aged) months of age were paced and instrumented for determination of developed left ventricular pressure. Necrosis was determined with triphenyltetrazolium. In the absence of preconditioning, young and aged adult hearts made globally ischemic for 45 min developed necrosis involving 53+/-6% and 49+/-6% of the myocardium, respectively. Contractile function (+dP/dt(max)) at 90 min of reperfusion was depressed by 80% in young and 52% in aged hearts, compared to values obtained prior to preconditioning. Preconditioning with two 5 min ischemia/5 min reperfusion cycles significantly reduced necrosis development and enhanced reperfusion contractile function in young hearts. However, in aged adult hearts, the preconditioning did not significantly reduce the development of necrosis or enhance reperfusion contractile function. These data suggest that aging reduces the effectiveness of preconditioning in providing cardioprotection against ischemic-induced myocardial necrosis.

"Preconditioning at a distance" in the isolated rabbit heart.

OBJECTIVE: Brief myocardial ischemia evokes a cardioprotective response, referred to as "ischemic preconditioning" (IP), that limits injury caused by a subsequent prolonged ischemic insult. The myocardial IP effect can be induced by ischemia of "distant" cardiac and noncardiac tissue, implicating the involvement of an as-yet-unidentified humoral trigger. If a preconditioning hormone exists, the authors hypothesize that the IP effect should be transferable, via administration of coronary effluent, from a preconditioned donor heart to a virgin non-preconditioned acceptor heart. METHODS: Isolated buffer-perfused rabbit hearts were assigned to one of four treatment groups in a donor/acceptor sequence. Donor hearts underwent either three IP cycles or a matched period of uninterrupted perfusion (control donors). Coronary perfusate collected from IP and control donor hearts was reoxygenated and transfused to virgin acceptor hearts. All hearts then underwent 30 minutes of global ischemia followed by 30 minutes of reperfusion. Left ventricular developed pressure (LVDP) (the authors' index of cardioprotection) was monitored throughout the protocol by a left ventricular (LV) balloon. RESULTS: In donor controls, LVDP assessed at 30 minutes post-reflow was restored to only 49 +/- 5% of baseline values. Recovery of LV function was significantly enhanced in both IP donor hearts (69 +/- 4%*) and IP acceptor hearts (70 +/- 6%*) vs donor controls (*p < 0.05), while, in acceptor controls, intermediate values of LVDP (62 +/- 7%) were obtained. CONCLUSION: The IP effect can be transferred between rabbit hearts, suggesting the presence of a humoral trigger signal for distant preconditioning. Isolating this hormone may have therapeutic and diagnostic implications in the management of acute myocardial ischemia.

Rabbit heart can be "preconditioned" via transfer of coronary effluent.

Brief myocardial ischemia not only evokes a local cardioprotective or "preconditioning" effect but also can render remote myocardium resistant to sustained ischemia. We propose the following hypotheses: remote protection is initiated by a humoral trigger; brief ischemia-reperfusion will result in release of the humoral trigger (possibly adenosine and/or norepinephrine) into the coronary effluent; and transfer of this effluent to a virgin acceptor heart will elicit cardioprotection. To test these concepts, effluent was collected during normal perfusion from donor-control hearts and during preconditioning ischemia-reperfusion from donor-preconditioned (PC) hearts. After reoxygenation occurred and aliquots for measurement of adenosine and norepinephrine content were harvested, effluent was transfused to acceptor-control and acceptor-PC hearts. All hearts then underwent 40 min of global ischemia and 60 min of reperfusion, and infarct size was delineated by tetrazolium staining. Mean infarct size was smaller in both donor- and acceptor-PC groups (9% of left ventricle) than in donor- and acceptor-control groups (36% and 34%; P < 0.01). Protection in acceptor-PC hearts could not, however, be attributed to adenosine or norepinephrine. Thus preconditioning-induced cardioprotection can be transferred between rabbit hearts by transfusion of coronary effluent. Although adenosine and norepinephrine are apparently not responsible, these results suggest that remote protection is initiated by a humoral mechanism.