NO-system is proposed as endogenous cardioprotectant and antifibrillatory factor [1,2]. Diuretic-hypokalemia is an increasingly common cause of arrhythmias [3]. Therefore, we attempted to define hypokalemia and/or hypokalemia disturbances as particular disturbances related to NO-system and possible therapeutic value of NOsystem- related agents: NO-synthase (NOS)-blocker, L-NAME, NOSsubstrate, L-arginine, and, particularly, stable gastric pentadecapeptide BPC 157 [4-6]. In this, we focused on so far not demonstrated with L-NAME/L-arginine application [1,2] NO-system-relations in mortal furosemide-diuresis-hypokalemia course; -ECG changes, carefully analyzed in rats [7] (prolongation of P, R, S, T waves and PR, RR, QRS, QT interval duration, and reduced amplitude of R, S, T wave (although not completely related to hypokalemia) [7]); -arrhythmias (AV conduction block, ventricular premature beats, ventricular tachycardia); -skeletal muscle myoclonus [8]; and -lethality 90-150 min post-furosemide. Likely, this furosemide-hypokalemia-NO-systemsyndrome should be worsened by L-NAME, ameliorated by L-arginine and finally, counteracted by BPC 157. Namely, BPC 157 interacts with NO-systems in prevention of chronic heart failure, reversal of already established chronic heart failure [9], preventing digitalis arrhythmias and counteracting already advanced digitalis arrhythmias [10], both in vivo and in vitro, always overriding the L-arginine’s cardioprotectant and antiarrhythmic effect [10-14]. Since the methyldigoxin toxicity depends particularly on hypokalemia [1,2,15], an analogous life saving effect of BPC 157 would occur in hypokalemic rats overloaded with a huge dose of furosemide. Of note, probably also important for expected cardioprotectant and antiarrhythmic effectiveness [9,10], BPC 157 (particularly effective in the whole gastrointestinal tract (GEPPPGKPADDAGLV, M.W. 1419, in trials for inflammatory bowel disease), wound treatment [4-6], stable and not degraded in human gastric juice (more than 24h)) represents a well-matched anti-ulcer peptide [4-6]. Producing no reported toxicity, it is effective alone without carrier [4-6], positively targeting particularly vasculature (i.e., removal of vascular clot) [16] and recently, reducing bleeding time and thrombocytopenia after heparin, warfarin or aspirin [17]. Thus, using furosemide over-load without potassium intravenous supplementation in rats as a background in present study (life treating hypokalemia (<2.7 mEq/L)), BPC 157 was applied intraperitoneally or intragastrically, alone or in combination with L-NAME and/or L-arginine. Medication was given prophylactically before furosemide or alternatively, as a therapy, after furosemide, when hypokalemia-third-degree AV block and ventricular tachycardia were already established. To prove possible general mechanism how BPC-157 could protect cells in hypokaliemic conditions we used HEK293 cell. Membrane voltages (V_m) of HEK293 cells were measured using the slow-whole-cell patch clamp technique [18].

Animals

Female Wistar Albino rats (190-220 g) randomly assigned (10 rats per each experimental group), were used (Medical Faculty, University of Zagreb). All experiments approved by the Local Ethics Committee.

Medication, ECG recording and assessment

The furosemide (Furosemid, Pliva) dose (100 mg/kg i.p.) that was used was considerably increased [7] to demonstrate that after such forced diuresis, there is an unfailing acute hypokalemic syndrome (serum potassium level (<3 mmol/L); ECG changes (prolonged P, R, S, T waves and PR, RR, QRS, QT interval duration along with reduced amplitude of R, S, T wave although not completely related to hypokalemia [7]; arrhythmias (AV block, ventricular premature beats, ventricular tachycardia) and skeletal muscles myoclonus presentation)) that would be invariably lethal without curative agent application. Pentadecapeptide BPC 157 (manufactured by Diagen, Ljubljana, Slovenia [4-6,9,10], (GEPPPGKPADDAGLV, M.W. 1419, partial sequence of human gastric juice protein BPC, peptide with 99% purity (HPLC, freely soluble in water at pH 7.0 and in saline) dissolved in saline or saline alone (5.0 ml/kg), were intraperitoneally or intragastrically given 15 minutes before the intraperitoneal injection of 100 mg/kg of furosemide (Edemid forte, Belupo) (prophylactic regimen). BPC 157 rats that received intraperitoneal or intragastrical application, received the dose of the 10 μg/kg b.w. or the dose of the 10 ng/kg b.w.. In addition, L-NAME (5 m/kg) and/or L-arginine (100 mg/kg) (and D-arginine (Sigma, St. Louis, MO, USA) were dissolved in saline as described before [10-14]) were given intraperitoneally. Alternatively, the described medication was given intraperitoneally or intragastrically at 90 minutes after furosemide (therapeutic regimen). In combination studies, L-NAME and/or L-arginine (since without effect, D-arginine was not further shown) were given with BPC 157 10 μg/kg intraperitoneally (L-NAME+BPC 157; L-arginine+BPC 157; L-NAME+L-arginine+BPC 157).

Generally, through the 90 min period after furosemide application, the rats were monitored (separate clinical assessment and urination (mL) in metabolic cage for rat (Ugo Basile) at 5, 15, 30, 45, 60, 75 and 90 min after furosemide).

At 90 min after furosemide, the rats were deeply anesthetized (Thiopental-Natrium Pulver 40 mg/kg i.p. plus diazepam 5 mg/kg i.p.), and the ECG was recorded continuously throughout the next 60 minutes in three main leads by positioning stainless steel electrodes on all four limbs, using an ECG monitor by Medtronic Analyser programmer (Minneapolis, MN, USA). The ECG assessment included analysis of wave (P, R, S, T) amplitude and duration, interval (PQ, QRS,RR, QT) duration, time (s) until the start of third-degree AV block, its duration (s), time (min) until onset of ventricular tachycardia and the duration (min) of ventricular tachycardia. Ventricular tachycardia was defined as a run of three or more premature QRS complexes, not to be defined in terms of its rate or the prevailing sinus rate [19]. Total atrioventricular block was defined when no supraventricular impulses conducted to the ventricles and consequently in electrocardiografphical finding P waves which reflect a sinus node rhythm were independent from QRS complexes.

In the studies of the agents effect on already established severe hypokalemia disturbances, 90 min after the furosemide challenge, significant hypokalemia was recognized by ECG signs (reduction of R, S, T wave amplitude, prolongation of P, R, S, T wave duration as well as PQ, RR, QT interval duration along with third-degree AV block and/or ventricular tachycardia appearance) and ECG changes were monitored until the end of the next 60 min period (which however, could not be further extended because of the fatal outcome, regularly occurring at that time).

In a separate group of rats, blood samples were collected using a heparin tube from the rat eye before furosemide challenge, and thereafter at the time of recognized hypokalemia by ECG signs at 90 min and at the end of 60 min-observation period (i.e., 150 min after furosemide challenge). The concentrations of plasma electrolytes were measured by an autoanalyzer (Olympus AU2700 analyzer).

The duration of myoclonus was accordingly assessed. Myoclonus is defined as sudden, brief, shock-like, involuntary movements caused by muscular contraction or inhibitors [20].

The time until death and the number of fatal outcomes (assessed by ECG, asystolia and lack of breathing for more than 3 minutes) in hypokalemia-induced arrhythmias was also assessed.

Cell culture

HEK293 cells were grown on glass coverslips in Dulbecco’s Modified Eagle Medium (DMEM) which contained 3.7 g/l NaHCO₃ in addition of 2 mM L-glutamine, 10 ml/l Penicilin/Streptomicin (10000E/10000 mg/ml) and 10% fetal calf serum (FCS). Cells were kept at 5% CO₂ at 37°C and used from experiments at passage 190, 5-6 days after trypsination (0.25% trypsin-EDTA solution, Sigma-Aldrich in Mg²⁺, Ca²⁺ free HANKS solution).

Patch clamp studies

Coverslips with HEK293 cells were mounted as the bottom of a perfusion chamber on an inverted microscope (Axiovert 10 Zeiss, Gottingen, Germany). Membrane voltages (V_m) of HEK293 cells were measured using the slow-whole-cell patch clamp technique [18]. A solution containing 145 mM NaCl, 1.6 mM K₂HPO₄, 0.4 mM KH₂PO₄, 5 mM D-glucose, 1 mM MgCl₂, 1.3 mM calcium gluconate (pH 7.4) was used in experiments. All experiments were performed at 37°C with a bath perfusion rate of 10 ml/min. Patch clamp pipettes were filled with 95 mM potassium gluconate, 30 mM KCl, 4.8 mM Na₂HPO₄, 1.2 mM NaH₂PO₄, 5 mM D-glucose, 1.3 mM calcium gluconate, 1.03 mM MgCl₂, and 1 mM ATP (pH 7.2). To this solution 160 μM nystatin were added to permeabilize the membrane under the pipette. Pipette resistance prepared this way had resistance of 5-10 MΩ Vm was measured with a patch-clamp amplifier (U. Frobe, Physiologische Institut, Freiburg, Germany) and recorded continuously on a pen recorder (WeKa graph, Kaltbrunn, Switzerland). All effects were analysed after V_m had reached a new steady state value and were compared to their averaged pre- and post control values.

To determine effects of BPC-157 on V_m of HEK293 cells during hypokalemic condition (hypokaliemic step), we used following solution: 145 mM NaCl, 1.6 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 5 mM D-glucose, 1 mM MgCl₂, 1.3 mM calcium gluconate (pH 7.4). This solution has potassium concentration of 0.4 mM which is hypokalemic compared to control solution ([K⁺] = 3.6 mM). After first hypokaliemic step (pre-control), cells were overperfused with BPC-157 (1 μM) for at least 2 min. Hyperkalemic step was repeated together with BPC-157 in the bath solution. After washing BPC-157 for few minutes, additional hypokalemic step was performed (post-control).

Statistical analysis

Statistical analysis was performed using parametric two-way mixed model ANOVA (one factor is repeated-measures) and Student Newman–Keuls test to compare the difference between groups. Fisher’s exact probability test was used to assess the number of dead and surviving rats. A P value of 0.05 or less was considered statistically significant.

Plasma potassium and other electrolyte concentrations; furosemide induced diuresis

Always, after furosemide overdose, steady severe hypokalemia (<2.7 mmol/L) (Tables 1A and 1B) is equally persisting, even after all agents averted to some extent furosemide-forced diuresis. In particular, L-arginine, BPC 157 postponed furosemide-diuresis. L-NAME showed a decrease of furosemide-diuresis, antagonized by L-arginine or BPC 157 (Tables 2A and 2B).

Table 1A: Electrolytes plasma concentrations (mmol/L), means ±SEM. BPC 157 administered intraperitoneally or intragastrically was given prophylactically before furosemide (100 mg/kg intraperitoneally) or alternatively, as a therapy, after furosemide, when hypokalemia- third-degree AV block and ventricular tachycardia were already established. * P<0.05, at least vs. initial presentation (italic) without furosemide.

Table 1B: Electrolytes plasma concentrations (mmol/L), means ±SEM. L-NAME and L-arginine administered intraperitoneally prophylactically before furosemide (100 mg/kg intraperitoneally) or alternatively, as a therapy, after furosemide, when hypokalemia- third-degree AV block and ventricular tachycardia were already established. *P<0.05, at least vs. initial presentation (italic) without furosemide.

Table 2A: Effects of BPC 157 on furosemide (100 mg/kg intraperitoneally)-diuresis (mL). Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. *P<0.05 significantly different from control values.

Table 2B: Effects of BPC 157 (10 μg/kg i.p.), L-NAME (5 mg/kg i.p.) and L-arginine (100 mg/kg i.p.) on furosemide (100 mg/kg i.p.)-diuresis (mL). Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. *P<0.05 significantly different from control values.

Severe acute hypokalemia syndrome (control rats presentation)

All hypokalemic rats consistently exhibited prolongation of the PQ, QRS, QT (Tables 3A, 3B, 4A and 4B), RR (Tables 5A and 5B) interval and all wave (P, R, S, T) duration, with a concomitant decrease of the amplitude of all waves except the P wave (Tables 3A, 3B, 4A, 4B), considerable number of premature ventricular beats (per min), marked duration of ventricular tachycardia, quite extensive duration of 3°AV block (min) (Table 6), no termination of arrhythmias, lethality was absolute and fatal outcome invariably occurred at the end of the observation period (Tables 6, 7A and 7B). In particular, along with the above mentioned ECG changes, furosemide rats also developed AV conduction block (including third degree) in Hiss-Purkinje system (3^rd degree AV block with wide QRS complexes (duration: 30-33 msec vs normal range 15-17.5 msec) and low ventricular frequency (90- 120/min vs. normal range 320-500/min) and polymorphic ventricular tachycardia, frequency between 270-290/min with also wide QRS complexes (duration 30-33 msec) and clockwise rotation of electrical axis (Table 8)) and an appearance of abnormal ventricular rhythm (multifocal ventricular premature beats or polymorphic ventricular tachycardia („torsades de pointes“) (Table 8). Along with hypokalemiainduced ECG abnormalities, all control rats manifest myoclonus i.e., sudden, brief, shock-like, involuntary movements throughout the entire 60 min test period (i.e., 90-150 minutes after furosemide) (Table 9).

Table 3A: Effects of hypokalemia on amplitudes (0.01mV) of ECG waves, ECG wave duration and interval duration (msec). BPC 157 administered intraperitoneally was given prophylactically before furosemide (100 mg/kg intraperitoneally). Values are mean ± SEM. ¥ - Two-way ANOVAs with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. †P<0.05 significantly different from control values.

Table 3B: Effects of hypokalemia on amplitudes (0.01mV) of ECG waves, ECG wave duration and interval duration (msec). BPC 157 administered intraperitoneally as a therapy, after furosemide (100 mg/kg intraperitoneally), when hypokalemia- third-degree AV block and ventricular tachycardia were already established. Values are mean ± SEM. †P<0.05 significantly different from control values.

Table 4A: Effects of hypokalemia on amplitudes (0.01mV) of ECG waves, ECG wave duration and interval duration (msec). BPC 157 administered intragastrically was given prophylactically before furosemide (100 mg/kg intraperitoneally). Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership.†P<0.05 significantly different from control values.

Table 4B: Effects of hypokalemia on amplitudes (0.01mV) of ECG waves, ECG wave duration and interval duration (msec). BPC 157 administered intragastrically, as a therapy, after furosemide (100 mg/kg intraperitoneally), when hypokalemia- third-degree AV block and ventricular tachycardia were already established. Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. †P<0.05 significantly different from control values.

Table 5A: Effects of hypokalemia on RR interval duration (msec) during sinus rhythm, ventricular tachycardia, third-degree AV block. BPC 157 (intraperitoneally or intragastrically) was given prophylactically before furosemide (100 mg/kg intraperitoneally). Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership.†P<0.05 significantly different from control values.

Table 5B: Effects of hypokalemia on RR interval duration (msec) during sinus rhythm, ventricular tachicardia, third-degree AV block. BPC 157 (intraperitoneally or intragastrically) as a therapy, after furosemide (100 mg/kg intraperitoneally), when hypokalemia- third-degree AV block and ventricular tachycardia were already established. Values are mean ± SEM. ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. †P<0.05 significantly different from control values.

Table 6: When hypokalemia- third-degree AV block and ventricular tachycardia were already established, rats were monitored throughout the 60 min after application BPC 157 (intraperitoneally or intragastrically) as a therapy at 90 min after furosemide for a number of premature ventricular beats (per min), duration of ventricular tachycardia (min), duration of third-degree AV block (min), absolute termination of arrhythmias (min.), time till fatal outcome (min.), mortality rate (surviving/dead rats). Values are mean ± SEM. †P<0.05 significantly different from control values.

Table 7A: Development of furosemide-induced hypokalemic arrhythmias after application of pentadecapeptide BPC 157 (10 μg/kg i.p.), L-NAME (5 mg/kg i.p.), inhibitor of NO-synthase, and L-arginine (100 mg/kg i.p.), substrate of NO-synthase and NO-precursor – PROPHYLACTIC REGIMEN (medication given 15 min. before furosemide). +P≤0.05 at least vs. control.

Table 7B: The effect of pentadecapeptide BPC 157 (10 μg/kg i.p.), L-NAME (5 mg/kg i.p.), inhibitor of NO-synthase, and L-arginine (100 mg/kg i.p.), substrate of NOsynthase on the established furosemide-induced hypokalemic arrhythmias in the rat – THERAPEUTIC REGIMEN (Medication given 90 min. after furosemide). ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. + P≤0.05 at least vs. control. * P≤0.05 at least vs. baseline.

Table 8: The effect of pentadecapeptide BPC 157 (10 μg/kg i.p.), L-NAME (5 mg/kg i.p.), inhibitor of NO-synthase, and L-arginine (100 mg/kg i.p.), substrate of NOsynthase on the interference interventricular implementation in furosemide-induced hypokalemic arrhythmias in the rat – THERAPEUTIC REGIMENT (Medication given 90 min. after furosemide). PROPHYLACTIC REGIMEN (Medication given at 15 min before furosemide). ¥ - Two-way ANOVA with repeated measure on one factor (P<0.05) difference between measurements depends on group membership. + P≤0.05 at least vs. control. * P≤0.05 at least vs. baseline.

Table 9: Absolute termination of furosemide-induced hypokalemic arrhythmias and myoclonismus after application of pentadecapeptide BPC 157 (10 μg/kg i.p.), L-NAME (5 mg/kg i.p.), inhibitor of NO-synthase, and L-arginine (100 mg/kg i.p.), substrate of NO-synthase and NO-precursor. †P<0.05 at least vs. control.

Stable gastric pentadecapeptide BPC 157

All BPC 157-furosemide rats survived. In general, in all hypokalemic BPC 157 rats AV block and abnormal ventricular rhythm either did not appear (BPC 157 before furosemide) or in rats presenting with the third-degree AV block and ventricular tachycardia, sinus rhythm was completely restored within a few minutes after BPC 157. The myoclonus was either not exhibited (BPC 157 given before furosemide) or reversed within a very short period (BPC 157 given in rats with already advanced arrhythmias and myoclonus after furosemide) (minutes, means ± SEM) (i.e., 5.5 ± 0.5 (10 μg/kg i.p.), 9.5 ± 1.5 (10 ng/kg i.p.), 9.0 ± 1.5 (10 μg/kg i.g.), 11.0 ± 1.0 (10 ng/kg i.g.), (vs. controls P<0.05, at least)). Specifically, BPC 157 was fully effective intraperitoneally (Table 3A, Table 3B) or intragastrically (Tables 4A and 4B), alone (Table 3-6) or in combination with L-NAME and/or L-arginine (Tables 7A, 7B, 8, and 9), given prophylactically before furosemide (Tables 3A, 4A, 5A, 7A, 8 and 9) or alternatively, as a therapy, after furosemide (Tables 3B, 5B, 6B, 7B, 8 and 9), when hypokalemia-third-degree AV block and ventricular tachycardia were already established).

L-arginine

Given before or after furosemide as described, L-arginine attenuates arrhythmias and prevents a lethal outcome (Tables 7A, 7B and 8). L-arginine affected the extra-cardiac manifestations like it did the cardiac manifestations (Table 9).

L-NAME

Contrarily, given before or after furosemide, the effect was aggravation of arrhythmias and a more rapid lethal outcome (Tables 7A, 7B and 8). Also, L-NAME aggravated the extra-cardiac manifestations like it did the cardiac manifestations (Table 9).

Combinations (L-NAME+L-arginine; L-NAME+BPC 157; L-NAME+L-arginine +BPC 157; BPC 157+L-arginine)

Prophylactically (at health condition) as well as therapeutically (90 min after acute furosemide overdose) combining worsening (NOsynthase (NOS)-blocker, L-NAME) /amelioration (NOS-substrate, L-arginine) /nearly complete counteraction (BPC 157) lead to null mortality, cardiac/extra-cardiac manifestation rapidly eliminated (L-NAME+BPC 157; L-NAME+L-arginine+BPC 157 (thus, BPC 157 preserved beneficial effect, completely eliminated L-NAME harmful effect) ; L-arginine+BPC 157 (BPC 157 beneficial effect not potentiated)) (Tables 7A, 7B, 8 and 9). Alternatively, we noted mutual counteraction, mortality, cardiac/extra-cardiac manifestation like in controls (L-NAME+L-arginine) (Tables 7A, 7B, 8 and 9). Thereby, in summary, L-NAME>control>L-arginine>BPC 157, involves both early development, and late advanced hypokalemia-syndrome while hypokalemia was always the same.

Cell model

To prove possible general mechanism how BPC-157 could protect cells in hypokaliemic conditions we used HEK293 cell. Membrane voltages (V_m) of HEK293 cells were measured using the slow-wholecell patch clamp technique [18]. In hypokalemic conditions (0.4 mM) cells hyperpolarized for -6.1 ± 1.1 mV, n=5 (Figure 1). After first hypokalemic step, we add to the solution 1 μM BPC-157 which depolarized cells for 4.6 ± 1.6 mV, n=5. Repeating hypokaliemic step in the presence of BPC-157, cells did not hyperpolarized (3.1 ± 1.6 mV, n=5, Figure 1). After washing BPC-157 from bath solution, under hypokalemic conditions cells hyperpolarized again (Figure 1).

Figure 1: BPC-157 (1 uM) abolished hyperpolarizationsof HEK293 cells during hypokaliemicconditions. After washing BPC-157 out, hyperpolarizationsresponse on hypokaliemiarecovered. Data represent the mean ± SE with the number of experiments given in brackets (paired experiments). P < 0.05 compared effects of hypokaliemicconditions on the membrane voltages (V_m) with and without presence of BPC-157, ** P < 0.05 effects of hypokaliemiaon mafter washing BPC-157 out compared to changes of V_m under hyokaliemicconditionsin the presence of BPC-157

Possibly, these findings in severe acute hypokalemia syndrome in rats (life treating furosemide over-load without potassium intravenous supplementation, BPC 157, intraperitoneally or intragastrically, alone or in combination with L-NAME and/or L-arginine) might contribute to understanding of K+ homeostatis and on the myocardial conduction system and on the skeletal muscle electrical system, the role of the NO system in this K+ - related electrical activity. And importantly, before furosemide or alternatively, as a therapy, after furosemide, when hypokalemia-third-degree AV block and ventricular tachycardia were already established, the beneficial effects obtained suggest potentially effective therapeutic interventions (null mortality, BPC 157>L-arginine) for acute hypokalemia. L-NAME/Larginine mutual counteraction ascertains full accuracy for NO-system effects and involvement [21]. Furthermore, BPC 157 more powerful than L-arginine (i.e., arrhythmias and myoclonus elimination (BPC 157) vs. attenuation (L-arginine), particularly evident when each of them was combined with L-NAME), alike BPC 157>L-arginine lifesaving potential in doxorubicine-intoxication [9], methyldigoxinintoxication, methyldigoxin-L-NAME-intoxication [10] all together generalize the same life-saving dose regimen (μg-ng) and route of application (intraperitoneal, intragastrical), and prophylactic and therapy protocols; both early development, and late advanced stage of doxorubicine-, methyldigoxin-intoxication and hypokalemiasyndrome. Also, BPC 157>L-arginine, BPC 157 effects not exaggerated by that of L-arginine [10,13,14] may represent distinctive ways in NO-alternative rescuing system. Finally, to prove possible general mechanism how BPC-157 could protect cells in hypokalemic conditions we used HEK293 cell. Repeating hypokalemic step in the presence of BPC-157, cells did not hyperpolarized. After washing BPC- 157 from bath solution, under hypokalemic conditions cells were less hyperpolarized.

However, despite all these arguments, the same hypokalemia in all furosemide-groups while different outcome further question to how BPC 157, L-arginine and L-NAME specifically worked. This could be not simply explained by the regular furosemide action, inhibition of Na-K ATPase activity in the medullar thick ascending limb of Henle’s loop [7] or increased distal tubular fluid flow, or aldosterone, catecholamine and antidiuretic hormones actions that could contribute to the increase in potassium secretion whatever they are working maximally, or were already exhausted. But, any feedback from the renal part, less likely to have direct influence (i.e., generally averted furosemide-forced diuresis, decreased by L-NAME, postponed by L-arginine and BPC 157) should be NO-system related (L-NAME is antagonized by L-arginine or BPC 157).

Furthermore, “same hypokalemia”-response could be discriminative. With accelerated death, it requires less diuresis in L-NAME-furosemide rats, NOS-blockade and disable NO-system, L-NAME-furosemide rats being more susceptible to hypokalemia and hypokalemia-cardiac and extra-cardiac disturbances; while with no mortality of L-arginine- and BPC 157-rats could sustain more diuresis with considerably better outcome. For instance, AV block and abnormal ventricular rhythm either did not appear (i.e., BPC 157+furosemide) or sinus rhythm completely rescued within a few minutes (i.e., furosemide+BPC 157)) provide a normal ECG presentation and survival despite steady severe hypokalemia in furosemide-rats. Counteraction of AV blocks (including third-degree AV block) occurs no matter what the total absence of AV conduction (in digitalis-intoxication [10] or AV conduction block (including third-degree AV block) was in Hiss-Purkinje system in furosemide-hypokalemic rats. Note, in hypokalemia, decrease of conduction velocity and prolongation of relative refractory period are more expressed in Hiss-Purkinje fibers than in ventricular fibers along with commonly thought hypokalemiainduced abnormal pacemaker activity attributed to increased slope of diastolic depolarization in Purkinje fibers [22,23].

Thus, besides the counteraction of hypokalemic disturbances in vitro, and thereby a direct effect on membrane, the “same hypokalemia”- question may be solved via targeting cardiac directly (i.e., BPC 157’s beneficial effect on myocardial muscle and conductive tissue was suggested also in doxorubicine- and methyldigoxin-intoxication) [9,10] or vasculature. Obviously, that effect should be multifactorial and different from the standard antiarrhytmic agents [9,10]. For instance, BPC 157 therapy likely involves a counteraction of downregulation of IKr potassium current (no consequence of the reduced extracellular potassium-paradoxical inhibition of the activity of the IKr potassium current [24] and delayed ventricular repolarization), counteracted suppression of conduction (counteracted prolongation of the PQ and QRS, RR interval), and increased refractoriness also counteracted (note, counteraction of prolongation of QT interval). Further - since no prolongation of the QT interval, and no abnormal ventricular rhythm appears, - variform ventricular tachycardia (“torsades de pointes“) also antagonized means counteraction of early after depolarization and resultant triggering (thought to cause torsades de pointes syndrome [25,26]. Besides, the decrease of ECG wave amplitude (which does not necessarily mean that conduction is suppressed, and is not thought to be related to the degree of hypokalemia [7] was also counteracted. Note, BPC 157 beneficial effect [9,10] also includes hypoxic injury and reoxygenation arrhythmias [27]. What’s more, presenting that hypokalemia [28] causes endothelial dysfunction and low NO levels, it is worth mentioning that BPC 157 maintains endothelium integrity [16,17] along with its effect on the NO-system and antagonization of LNAME effect [14]. Besides, although not directly related, other BPC 157 effects may also be indicative to prevent hypokalemia-disturbances. For instance, hypokalemia is important in insulin resistance development [29] while BPC 157 may also counteract insulin resistance [30].

Providing that potassium is essential for many body functions, including muscle and nerve activity [31] and the same essential rescuing role of NO-system in both heart [1,2] and skeletal muscle [32] a supportive analogy noted in the severely hypokalemic furosemide-rats between cardiac and extra-cardiac manifestations (myoclonus) [8] presentation and therapy (L-NAME (aggravation) vs. L-arginine (attenuation), BPC 157 (elimination)) should be expected. This could be at the best perceived with respect to the analogous BPC 157 treatment effects; in prophylaxis and therapy (myoclonus either did not appear or severe myoclonus was very rapidly eliminated). Further, this analogy actually conveys in mentioned NO-system-terms cardiac muscle (decreased extracellular potassium: myocardial hyperexcitability with the potential to develop re-entrant arrhythmias) and skeletal muscle (decreased potassium levels in the extracellular space: hyperpolarization of the resting membrane potential, a greater than normal stimulus for depolarization of the membrane in order to initiate an action potential) [24]. In support, presenting the general significance of skeletal muscle for plasma potassium homeostasis [31], BPC 157’s heart effect [9,10,27] are along with prominent neuroprotective effect [33,34], marked increase of the healing rate of severely damaged skeletal muscles and recovering of their function [35-37].

In summary, for potassium-related disturbances and BPC 157 antiarrhythmic and recovering effects, not only in theory, to obtain a successful recovery, besides interaction with NO-system that was now demonstrated [10-14], it could be essential to modify the negative chain of events. These include; the prematurely depolarized membrane, early after derpolarization, prolonged cardiac repolarization when extracellular K+ is low which provides polymorphic ventricular tachycardias and a long QT interval (i.e., “torsades de pointes”), which were all antagonized, are both, prevented (i.e., prophylactic regimens) and reversed (therapeutic regimens) by a single BPC 157 i.p./i.g. administration. Thus, no supplementation of potassium and/or cessation of the medications that induce hypokalemia clearly suggest therapeutic significance in acute hypokalemia for null mortality, BPC 157>L-arginine evidenced in this study.

This work is supported by the research grant of Ministry of Science Education and Sports of Republic of Croatia.