Development of critical thinking skills in human anatomy and physiology.

Application-of-knowledge skills are highly valued in clinical medicine, as indicated by recent changes to licensure and entrance exams for nursing and physician programs (i.e., the NCLEX and MCAT). Such emphasis should be both welcomed and supported by approaches to teaching human anatomy and physiology that emphasize critical thinking skills built upon logic, reasoning, and judgment. The argument for development of these skills is not simply philosophical. Rather, such emphasis is strongly supported by a 2016 Johns Hopkins study (Makary MA, Daniel M. BMJ 353: i2139, 2016) that estimates that medical errors are now the third leading cause of death in the United States! Active learning techniques known to require critical thinking skills are often supplemental to standard expository lecturing or other avenues of imparting content knowledge (reading, videos, etc.). We propose that all content dissemination can and should provide for the development of critical thinking skills, preparing students for active learning techniques requiring this ability. This can be accomplished by establishing an intellectual framework for understanding the adaptive benefits of anatomical or physiological traits. Additionally, explanations conveying the causality of mechanistic sequences result in learning content within intuitive functional groups rather than as isolated phenomena, the latter often accomplished mainly through memorization as opposed to real understanding. Here, we provide a template for lecture development based upon these principles as well as a specific example from human anatomy and physiology. Our hope is to provide a model for how students should think about all physiology, making comprehensive coverage of content (an impossible task!) much less important.NEW & NOTEWORTHY Critical thinking skills are essential to the effective performance of many careers, particularly those involving health care. To aid the development of these skills in physiology, the formation of logical cognitive frameworks needs to be supported via instruction that emphasizes the context of physiological functions (the "why") as well as the causality of their sequential actions. Within such frameworks, students become capable of cognitive reasoning required to reach intuitive conclusions after system perturbations.

Vasa recta voltage-gated Na+ channel Nav1.3 is regulated by calmodulin.

Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na(+) (Na(V)) conductance. We examined expression of Na(V) isoforms in DVR and tested for regulation of Na(V) currents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only Na(V)1.3. Immunoblot of outer medullary homogenate verified Na(V)1.3 expression, and fluorescent immunochemistry showed Na(V)1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na(V)1.3 is confined to alpha-smooth muscle actin-positive vascular bundles. Na(V)1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na(V)1.3 or a GST-Na(V)1.3 COOH-terminal fusion protein. In patch-clamp experiments, Na(V) currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte Na(V) currents; however, raising electrode free Ca(2+) from 20 to approximately 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na(V)1.3C was not affected by Ca(2+) concentration. We conclude that Na(V)1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca(2+). Inhibition of CaM binding suppresses pericyte Na(V) currents.

Descending vasa recta endothelium is an electrical syncytium.

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10-30 ms. Carbenoxolone (100 microM) and 18beta-glycyrrhetinic acid (18betaGRA; 100 microM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] was monitored approximately 200 microm away. A 40-mV endothelial depolarization was accompanied by a 26.1 +/- 5.5-mV change in DiBAC(4)(3) fluorescence. DiBAC(4)(3) fluorescence did not change after 18betaGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca(2+) responses arising from mechanical perturbation of the DVR wall were attenuated by 18betaGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.

KATP channel conductance of descending vasa recta pericytes.

Using nystatin-perforated patch-clamp and whole cell recording, we tested the hypothesis that K(ATP) channels contribute to resting conductance of rat descending vasa recta (DVR) pericytes and are modulated by vasoconstrictors. The K(ATP) blocker glybenclamide (Glb; 10 microM) depolarized pericytes and inhibited outward currents of cells held at -40 mV. K(ATP) openers pinacidil (Pnc; 10 microM) and P-1075 (1 microM) hyperpolarized pericytes and transiently augmented outward currents. All effects of Pnc and P-1075 were fully reversed by Glb. Inward currents of pericytes held at -60 mV in symmetrical 140 mM K(+) were markedly augmented by Pnc and fully reversed by Glb. Ramp depolarizations in symmetrical K(+), performed in Pnc and Pnc + Glb, yielded a Pnc-induced, Glb-sensitive K(ATP) difference current that lacked rectification and reversed at 0 mV. Immunostaining identified both K(IR)6.1, K(IR)6.2 inward rectifier subunits and sulfonurea receptor subtype 2B. ANG II (1 and 10 nM) and endothelin-1 (10 nM) but not vasopressin (100 nM) significantly lowered holding current at -40 mV and abolished Pnc-stimulated outward currents. We conclude that DVR pericytes express K(ATP) channels that make a significant contribution to basal K(+) conductance and are inhibited by ANG II and endothelin-1.

Myocardial reperfusion injury: etiology, mechanisms, and therapies.

Reperfusion of ischemic myocardium is required for tissue survival; however, reperfusion elicits pathologic consequences. Myocardial reperfusion injury is a multifarious process that is mediated in part by oxygen free radicals, neutrophil-endothelium interactions, apoptosis, and intracellular calcium overload. The oxygen paradox describes the contradictory need to delivery oxygen to ischemic tissue and the resultant reduction of oxygen to form free radicals that are involved in macromolecule oxidation, membrane disfunction, apoptosis, and damaged calcium sequestering ability, which results in hypercontracture. These cell-damaging crises are amplified by the excessive activation of neutrophils, which promote the formation of proinflammatory mediators, oxygen radicals, and the reduction of endothelial nitric oxide formation, leading to increased neutrophil-endothelium interactions and capillary occlusion. Neutrophil action is twofold, however, because it is required for necrotic debris removal after severe ischemia. The oxygen radicals produced by neutrophils, endothelium, and myocytes may also play a role in activating the apoptotic cascade. Although the role of apoptosis in reperfusion injury is controversial, apoptotic cells are found in infarcted tissue. One of the key mediators may be increased inner mitochondrial membrane permeability, resulting in reduced ATP formation, release of cytochrome c, and caspase activation, which is key to promotion of apoptosis. Increased mitochondrial membrane permeability occurs during exposure to supraphysiological calcium concentrations. This occurs because of compensatory Na+/Ca2+ exchange to remove the excess intracellular sodium resulting from decreased Na+/K+ pumping during ischemia and increased Na+/H+ exchange following reperfusion. Supraphysiological calcium elicits hypercontracture and cellular damage. The various therapies being developed to diminish myocardial reperfusion injury involve inhibition of the processes described above as well as others. Although single therapies have shown some promise, the complexity of the response to reperfusion has made dramatic improvement elusive. Effective treatment will most likely require multifaceted antagonism of the numerous pathological cascades initiated by reperfusion.

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling.

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.

Angiotensin II constriction of rat vasa recta is partially thromboxane dependent.

We tested the hypothesis that thromboxane generation mediates vasoconstriction of isolated outer medullary descending vasa recta (OMDVR) by angiotensin (Ang) II. The lipoxygenase and cyclooxygenase (COX) inhibitor eicosatetraynoic acid (1 micromol/L) and the COX inhibitor indomethacin (1 micromol/L) partially reversed Ang II (1 nmol/L) constriction of in vitro perfused OMDVR. To determine whether thromboxane is a mediator of Ang II-induced vasoconstriction, a thromboxane synthase inhibitor, U63577A (1 micromol/L), and thromboxane receptor antagonists, SQ-29548 or BMS-180,291 (1 micromol/L, each), were introduced into the bath of vessels that had been preconstricted by Ang II (1 nmol/L). These agents significantly inhibited vasoconstriction induced by Ang II. In contrast, SQ-29548 and U63557A did not affect vessels preconstricted by raising extracellular KCl from 5 to 100 mmol/L. The thromboxane receptor agonist U46619 (1 micromol/L) constricted OMDVR, an effect that was blocked by the antagonist BMS-180,291. In separate protocols, microperfused OMDVR were pretreated with U63577A or SQ-29548, after which they were exposed to luminal Ang II to induce vasoconstriction. Both agents inhibited vasoconstriction whether preexposure to them was via the bath or the perfusate. We conclude that Ang II-induced constriction of OMDVR is partly mediated by metabolites of arachidonic acid, including thromboxanes.