Abatement of Forskolin-induced Intestinal Fluid Secretion by Modulation of Intracellular Camp With Penicillin

Millions of people die every year due to diarrheal related diseases, with infants and the elderly making up the majority of these deaths. Deaths are caused by excessive intestinal uid and electrolyte secretion and are especially common in impoverished developing countries. Antibiotics have been classically used as a method to treat diarrhea-related pathologies by modulating the gut microbiome. We recently reported that penicillin may protect against disease-induced excessive uid and electrolyte secretion via a genetics-independent, microbiome-independent mechanism in individual colonic crypt cells. In this study we investigated whether microbial-independent protective effects of penicillin against uid secretion can be observed in the rat small intestine at the whole-tissue level. Here we report that penicillin has a signicant dose-dependent protective effect against uid secretion in induced models of diarrhea in the microbiome decient rat small intestine. Penicillin can rapidly bring uid secretion down to levels comparable to healthy controls. Our results suggest, for the rst time, an alternative function for penicillin G as a cost-effective and fast-acting treatment against diarrheal symptoms without dependence on modulating the behavior of the existing gut microbiome.

As an antimicrobial, penicillin is a beta-lactam antibiotic containing a four-membered beta-lactam ring which blocks the activity of transpeptidases which are enzymes that form peptide cross-linkages in bacteria [9,10]. Previous studies demonstrated that Penicillin can increase intracellular cyclic nucleotide levels in mammals [11]. We recently reported that members of the Penicillin family function in a microbiome-independent manner to activate H/K ATPase in isolated rat colonic crypt cells by possibly modulating the cAMP pathway [12,13]. Due to the ability of H/K ATPase to uptake potassium, Penicillin may provide a potassium rescue mechanism that prevents life-threatening electrolyte and uid losses.
Although antibiotics are associated with diarrhea by altering host microbiome, Penicillin may trigger a compensatory response to reabsorb electrolytes in the colon [13]. However, whether Penicillin offers similar protective effects in the mammalian small intestine is still unknown. Previous reports elucidated that forskolin (FSK), the rst main labdane diterpenoid isolated from the Indian plant Plectranthus barbatus Andrews, increases intracellular cAMP and can in turn affect transmembrane ion transport [3,14]. Thus, FSK-induced cellular uid secretion has been used as a model for studying secretory diarrhea [15]. In this study, we used FSK to mimic diarrheal conditions in the rat small intestine.
Here, we report penicillin's ability to abate FSK-induced intestinal uid secretion in the rat small intestine.
Thus, there is use for penicillin, given acutely, to treat diarrheal disordersas a fast-response short-term alternative at the beginning of the therapeutic window to quickly modulate excessive intestinal uid secretion. Results:

Modeling Diarrheal Conditions with Forskolin in the Small Intestine
To determine the intraluminal uid secretion under control conditions in the intestinal segments we examined the change in FITC-Inulin concentration in the lumen in the absence of FSK or penicillin G (PenG). Under these conditions there was a 17.67% decrease in FITC-Inulin concentration over the period of 80 min (Table 1, Fig. 1). The tted slope of this decrease was − 0.2032 µM FITC-Inulin/min. This re ects the resting or basal level of uid secretion (control). The uorescence decreases as it is diluted with uid secreted into the lumen. We next examined the effect of adding FSK to the intraluminal perfusate. The addition of FSK alone resulted in a ~ 35% decrease in FITC-Inulin concentration at 80 min with a slope of -0.5495 µM/min ( Fig. 2A, Table 1). This re ects the increased uid secretion elicited by FSK. FSK stimulated uid secretion was signi cantly greater with almost a2-fold decrease in FITC-Inulin concentration compared to control conditions (34.92% vs 17.67%) ( Table 1 and Fig. 1b).   Table 1). In the presence of 5.0 mM PenG (our maximum dose) we observed a 16.95% decrease at -0.2133 µM/min in FITC-Inulin concentration (Fig. 2a, Fig. 2b, Table 1). This dose ( Table 1. We also analyzed the decrease in uorescence as a function of PenG concentration. This is shown in Fig. 4 where we plotted the data in a nonlinear t (inhibitor vs. response curve) using GraphPad. The IC 50 of PenG is calculated from the Prism analysis t to be 0.18 mM. The linear line in Fig. 4 is drawn to demonstrate the control level in the decrease in FITC-inulin with application of FSK and no PenG. Thus, PenG is able to counteract the induced uid secretion produced with FSK. This would indicate that PenG could be useful to reduce the excess uid secretion observed in diarrheal diseases (Figs. 3 and 4).

Discussion:
Throughout the history of medicine, many medications have been found to have alternative uses outside their originally intended function. In the eld of Gastroenterology, current research has shown that the antibiotics azithromycin, daxomicin, levo oxacin, and cipro oxacin all have been deployed as alternative therapies for cases of acute watery diarrhea and dysentery. In this study, we show that penicillin G, another commonly prescribed antibiotic, also has the potential to be repurposed and used as a therapeutic measure against cases of diarrhea and dysentery. A major consideration is also the cost savings using penicillin provides. A quick check of pharmacy prices shows azithromycin cost about $1.50 per tablet while penicillin is about $0.23 in the United States.
The breakthrough discovery of penicillin occurred in 1928 when it rst became widely recognized as the original modern antibiotic [16]. Penicillin originally found its use as a cure for various infectious diseases, such as bacterial endocarditis, meningitis, pneumococcal pneumonia, and streptococcal septicemia [9]. However, since then, penicillin is no longer being used to treat these infections due to the rapid spread of bacterial resistance to penicillin. Although penicillin isn't being widely utilized to treat bacterial infections, it has found new uses as a treatment for sexually transmitted diseases, and penicillin G currently remains the drug of choice for treating all stages of syphilis (Treatment of sexually transmitted diseases, 1986).
Our data suggests another off-label use for penicillin G for not only treating bacterial infections, but also as a treatment for diarrhea and dysentery. Our study using an ex-vivo perfusion device, demonstrates that the intraluminal application of penicillin G provides a protective effect on the distal small intestine against FSK induced uid secretion (mimicking diarrhea or dysentery). The signi cant effects of penicillin G perfusion as a means to abate the secretory effects of FSK suggest that this therapeutic may be used at the rst signs of secretory diarrhea. Penicillin G has the potential to be a low-cost and easily accessible short-term alternative for diarrheal diseases intended for use in developing countries.
At the moment, Cipro oxacin remains the most commonly used pharmaceutical in the prevention of diarrhea [17]. However, Cipro oxacin has been shown to possess arrhythmogenic properties and is linked to the development of cardiac arrhythmias and an increased risk of tears of cardiac vasculature [18]. This makes the potential of Penicillin G as an antidiarrheal a safer, low-risk alternative that can also be used as a treatment option for patients with pre-existing heart conditions, as it has no reported cardiac side effects, unlike present conventional antibiotic treatments.
Our study demonstrated that intraluminal application of penicillin G. provides a dose dependent protective effect on the distal small intestine against FSK induced uid secretion. Penicillin doses down to 0.5 mM in the presence of FSK prevented uid secretion with no statistical difference in uid secretion compared to a healthy intestinal segment perfused with control solutions. The counteracting effects of penicillin G on FSK exposed tissue indicate the antibiotic's ability to prevent excessive uid loss that is associated with exposure to FSK and cellular uid loss due to cAMP upregulation [14]. Our results suggest an alternative function for penicillin G as a cost-effective, rapid low-risk therapeutic measure against diseases with diarrhea related symptoms with the small and large intestines as potential for the extent of uid secretion. Penicillin G at various dosages were also added intraluminally to different experimental groups to observe the protective effects against FSK induced uid secretion. FSK was used to increase levels of cAMP within the cell and as seen in diarrheal diseases. Extraluminal perfusion contained warm HRBS alone. The pH and osmolality (mOsm) of all perfused solutions were adjusted to 7.4 and 300 ± 5 respectively at 37°C.

Experimental Conditions
In this study, we de ne an experiment as the harvest of the distal small intestine segment for use on our